Method for treating cancer using a combination of dna damaging agents and atr inhibitors

ABSTRACT

Described herein are methods of treating a proliferative disorder (e.g., cancer) in a patient by administering a DNA damaging agent and between about 12 and about 48 hours later administering to the subject a compound that inhibits ATR protein kinase. Methods of treating a proliferative disorder (e.g., cancer) in a patient by administering a compound that inhibits ATR protein kinase are also described. Exemplary ATR inhibitors are represented by Formulae A-I and A-II:or a pharmaceutically acceptable salt thereof, wherein the variables are as defined herein.

BACKGROUND OF THE INVENTION

Cancers as a group account for approximately 13% of all deaths each year with the most common being: lung cancer (1.4 million deaths), stomach cancer (740,000 deaths), liver cancer (700,000 deaths), colorectal cancer (610,000 deaths), and breast cancer (460,000 deaths). The three most common childhood cancers are leukemia (34%), brain tumors (23%), and lymphomas (12%). Rates of childhood cancer have increased by 0.6% per year between 1975 to 2002 in the United States and by 1.1% per year between 1978 and 1997 in Europe. This makes invasive cancer the leading cause of death in the developed world and the second leading cause of death in the developing world. Accordingly, there is a need to identify novel and efficacious therapeutic strategies that mitigate the limitations of current anti-cancer drugs.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the unexpected discovery that ATR inhibitors administered about 12-48 hours after DNA damaging agents are particularly effective at treating proliferative diseases. Accordingly, aspects of the disclosure relate to a method of treating a proliferative disorder, such as cancer, in a subject, the method comprising administering to a subject in need thereof a DNA damaging agent and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase.

In some embodiments, said DNA-damaging agent is selected from the group consisting of chemotherapy and radiation treatment. In some embodiments, said DNA-damaging agent is independently selected from the group consisting of ionizing radiation, radiomimetic neocarzinostatin, a platinating agent, a Topo I inhibitor, a Topo II inhibitor, an antimetabolite, an alkylating agent (e.g., alkyl sulphonate), and an antibiotic.

In some embodiments, said DNA-damaging agent is a platinating agent selected from the group consisting of Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, Lobaplatin, Triplatin Tetranitrate, Picoplatin, Satraplatin, ProLindac, and Aroplatin.

In some embodiments, said DNA-damaging agent is a Topo I inhibitor selected from the group consisting of Camptothecin, Topotecan, Irinotecan/SN38, Rubitecan, and Belotecan. In some embodiments, said DNA-damaging agent is a Topo II inhibitor selected from the group consisting of Etoposide, Daunorubicin, Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin, Zorubicin, and Teniposide.

In some embodiments, said DNA-damaging agent is an antimetabolite selected from the group consisting of Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, 6-Mercaptopurine, 5-Fluorouracil, Azacitidine, and Hydroxyurea.

In some embodiments, said DNA-damaging agent is an alkylating agent selected from the group consisting of Mechlorethamine, Cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Melphalan, Prednimustine, Bendamustine, Uramustine, Estramustine, Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide, Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin, nitrogen mustards, nitrosoureas, triazenes, alkyl sulfonates, Procarbazine, aziridines, and Plicamycin.

In some embodiments, said DNA-damaging agent is an antibiotic selected from the group consisting of Hydroxyurea, Anthracyclines, Anthracenediones, and antibiotics from the Streptomyces family.

In some embodiments, the proliferative disorder may be cancer, such as a solid tumor cancer. In some such embodiments, said cancer is a solid tumor cancer selected from the group consisting of lung cancer, gastrointestinal cancer, genitourinary tract cancer, liver cancer, bone cancer, nervous system cancer, gynecological cancer, skin cancer, thyroid gland cancer, and adrenal gland cancer.

In some embodiments, said cancer is a solid tumor cancer selected from the following cancers: Oral: buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestines (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillary thyroid carcinoma, follicular thyroid carcinoma; medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma; Adenoid cystic carcinoma; and Adrenal glands: neuroblastoma.

In some embodiments, said cancer is non-small cell lung cancer, small cell lung cancer, pancreatic cancer, biliary tract cancer, head and neck cancer, bladder cancer, colorectal cancer, glioblastoma, esophageal cancer, breast cancer, hepatocellular carcinoma, or ovarian cancer.

In some embodiments, said cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, and triple negative breast cancer.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-4     heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, wherein said monocyclic aryl or     heteroaryl ring is optionally fused to another ring to form an 8-10     membered bicyclic aryl or heteroaryl ring having 0-6 heteroatoms     independently selected from the group consisting of nitrogen,     oxygen, and sulfur; each R¹ is optionally substituted with 1-5 J¹     groups; -   R² is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-3     heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, wherein said monocyclic aryl or     heteroaryl ring is optionally fused to another ring to form an 8-10     membered bicyclic aryl or heteroaryl ring having 0-4 heteroatoms     independently selected from the group consisting of nitrogen,     oxygen, and sulfur; each R² is optionally substituted with 1-5 J²     groups; -   L is —C(O)NH— or —C(O)N(C₁₋₆alkyl)-; -   n is 0 or 1; -   each J¹ and J² is independently halo, —CN, —NO₂, —V¹—R, or     —(V²)_(m)-Q; -   V¹ is a C₁₋₁₀aliphatic chain, wherein 0-3 methylene units are     optionally and independently replaced with O, N″, S, C(O), S(O), or     S(O)₂; V¹ is optionally substituted with 1-6 occurrences of J^(V1); -   V² is a C₁₋₁₀aliphatic chain, wherein 0-3 methylene units are     optionally and independently replaced with O, N″, S, C(O), S(O), or     S(O)₂; V² is optionally substituted with 1-6 occurrences of J^(V2); -   m is 0 or 1; -   Q is a 3-8 membered saturated or unsaturated monocyclic ring having     0-4 heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, or a 9-10 membered saturated or     unsaturated bicyclic ring having 0-6 heteroatoms independently     selected from the group consisting of nitrogen, oxygen, and sulfur;     each Q is optionally substituted with 0-5 J^(Q); -   each J^(V1) or J^(V2) is independently halogen, CN, NH₂, NO₂,     C₁₋₄aliphatic, NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, OH,     O(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,     C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, NHCO(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)CO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic),     NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein     said C₁₋₄aliphatic is optionally substituted with halo; -   R is H or C₁₋₆aliphatic, wherein said C₁₋₆aliphatic is optionally     substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),     NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), CO(C₁₋₄aliphatic),     O(haloC₁₋₄aliphatic), or haloC₁₋₄aliphatic; -   each J^(Q) is independently halo, oxo, CN, NO₂, X—R, or —(X)_(p)-Q⁴; -   p is 0 or 1; -   X is C₁₋₁₀aliphatic, wherein 1-3 methylene units of said     C₁₋₆aliphatic are optionally replaced with —NR, —O—, —S—, C(O),     S(O)₂, or S(O); wherein X is optionally and independently     substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),     NO₂, CN, CO(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,     C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, SO(C₁₋₄aliphatic),     SO₂(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic), SO₂N(C₁₋₄aliphatic)₂,     NHC(O)(C₁₋₄aliphatic), N(C₁₋₄aliphatic)C(O)(C₁₋₄aliphatic), wherein     said C₁₋₄aliphatic is optionally substituted with 1-3 occurrences of     halo; -   Q⁴ is a 3-8 membered saturated or unsaturated monocyclic ring having     0-4 heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, or a 8-10 membered saturated or     unsaturated bicyclic ring having 0-6 heteroatoms independently     selected from the group consisting of nitrogen, oxygen, and sulfur;     each Q⁴ is optionally substituted with 1-5 J^(Q4); -   J^(Q4) is halo, CN, or C₁₋₄alkyl, wherein up to 2 methylene units     are optionally replaced with O, NR*, S, C(O), S(O), or S(O)₂; -   R is H or C₁₋₄alkyl wherein said C₁₋₄alkyl is optionally substituted     with 1-4 halo; -   R″ and R* are each independently H, C₁₋₄alkyl, or is absent, wherein     said C₁₋₄alkyl is optionally substituted with 1-4 halo;     wherein the proliferative disorder has one or more defects in the     ATM signaling pathway.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-1:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-I-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   Ring A is

-   J⁵o is H, F, Cl, C₁₋₄aliphatic, O(C₁₋₃aliphatic), or OH; -   J⁵p is

-   J⁵p1 is H, C₁₋₄aliphatic, oxetanyl, tetrahydrofuranyl,     tetrahydropyrany, wherein J⁵p1 is optionally substituted with 1-2     occurrences of OH or halo; -   J⁵p2 is H, methyl, ethyl, CH₂F, CF₃, or CH₂OH; -   J²o is H, CN, or SO₂CH₃; -   J²m is H, F, Cl, or methyl; -   J²p is —SO₂(C₁₋₆alkyl), —SO₂(C₃₋₆cycloalkyl), —SO₂(4-6 membered     heterocyclyl), —SO₂(C₁₋₄alkyl)N(C₁₋₄alkyl)₂, or —SO₂(C₁₋₄alkyl)-(4-6     membered heterocyclyl), wherein said heterocyclyl contains 1     heteroatom selected from the group consisting of oxygen, nitrogen,     and sulfur, and wherein said J²p is optionally substituted with 1-3     occurrences halo, OH, or O(C₁₋₄alkyl).

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-2:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-II:

or a pharmaceutically salt thereof, wherein:

-   R¹⁰ is fluoro, chloro, or —C(J¹⁰)₂CN; -   J¹⁰ is independently H or C₁₋₂alkyl; or -   two occurrences of J¹⁰, together with the carbon atom to which they     are attached, form a 3-4 membered optionally substituted carbocyclic     ring; -   R²⁰ is H, halo, —CN, NH₂, a C₁₋₂alkyl optionally substituted with     0-3 occurrences of fluoro, or a C₁₋₃aliphatic chain, wherein up to     two methylene units of the aliphatic chain are optionally replaced     with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); -   R³ is H, halo, C₁₋₄alkyl optionally substituted with 1-3 occurrences     of halo, C₃₋₄cycloalkyl, —CN, or a C₁₋₃aliphatic chain, wherein up     to two methylene units of the aliphatic chain are optionally     replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); -   R⁴ is Q¹ or a C₁₋₁₀aliphatic chain, wherein up to four methylene     units of the aliphatic chain are optionally replaced with —O—,     —NR^(a)—, —C(O)—, or —S(O)_(z)—; each R⁴ is optionally substituted     with 0-5 occurrences of J^(Q1); or -   R³ and R⁴, taken together with the atoms to which they are bound,     form a 5-6 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; the ring formed by R³ and R⁴ is optionally substituted     with 0-3 occurrences of J^(Z); -   Q¹ is a 3-7 membered fully saturated, partially unsaturated, or     aromatic monocyclic ring, the 3-7 membered ring having 0-3     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or an 7-12 membered fully saturated, partially     unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms     selected from the group consisting of oxygen, nitrogen, and sulfur; -   J^(Z) is independently C₁₋₆aliphatic, ═O, halo, or     O; -   J^(Q1) is independently-CN; halo; ═O; Q²; or a C₁₋₈aliphatic chain,     wherein up to three methylene units of the aliphatic chain are     optionally replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z)—; each     occurrence of J^(Q1) is optionally substituted by 0-3 occurrences of     J^(R); or -   two occurrences of J^(Q1) on the same atom, taken together with the     atom to which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; wherein the ring formed by two occurrences of J^(Q1) is     optionally substituted with 0-3 occurrences of J^(X); or -   two occurrences of J^(Q1), together with Q¹, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   Q² is independently a 3-7 membered fully saturated, partially     unsaturated, or aromatic monocyclic ring having 0-3 heteroatoms     selected from the group consisting of oxygen, nitrogen, a sulfur; or     an 7-12 membered fully saturated, partially unsaturated, or aromatic     bicyclic ring having 0-5 heteroatoms selected from the group     consisting of oxygen, nitrogen, and sulfur; -   J^(R) is independently —CN; halo; ═O;     O; Q³; or a C₁₋₆aliphatic chain, wherein up to three methylene units     of the aliphatic chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z)—; each J^(R) is optionally substituted with 0-3     occurrences of J^(T); or -   two occurrences of J^(R) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; wherein the ring formed by two occurrences of J^(R) is     optionally substituted with 0-3 occurrences of J^(X); or -   two occurrences of J^(R), together with Q², form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   Q³ is a 3-7 membered fully saturated, partially unsaturated, or     aromatic monocyclic ring having 0-3 heteroatoms selected from the     group consisting of oxygen, nitrogen, and sulfur; or an 7-12     membered fully saturated, partially unsaturated, or aromatic     bicyclic ring having 0-5 heteroatoms selected from the group     consisting of oxygen, nitrogen, and sulfur; -   J^(X) is independently —CN; ═O; halo; or a C₁₋₄aliphatic chain,     wherein up to two methylene units of the aliphatic chain are     optionally replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z)—; -   J^(T) is independently halo, —CN;     O; ═O; —OH; a C₁₋₆aliphatic chain, wherein up to two methylene units     of the aliphatic chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z)—; or a 3-6 membered non-aromatic ring having     0-2 heteroatoms selected from the group consisting of oxygen,     nitrogen, and sulfur; each occurrence of J^(T) is optionally     substituted with 0-3 occurrences of J^(M); or -   two occurrences of J^(T) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or -   two occurrences of J^(T), together with Q³, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   J^(M) is independently halo or C₁₋₆aliphatic; -   z is 0, 1 or 2; and -   R^(a) is independently H or C₁₋₄aliphatic.

In some embodiments, R¹ and R³ are fluoro. In some embodiments, R⁴ is Q¹. In some embodiments, Q¹ is independently piperidinyl and imidazolyl.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-3:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-II-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹⁰ is fluoro, chloro, or —C(J¹⁰)₂CN; -   J¹⁰ is independently H or C₁₋₂alkyl; or -   two occurrences of J¹, together with the carbon atom to which they     are attached, form an optionally substituted 3-4 membered     carbocyclic ring; -   R³ is independently H, chloro, fluoro, C₁₋₄alkyl optionally     substituted with 1-3 occurrences of halo, C₃₋₄cycloalkyl, —CN, or a     C₁₋₃aliphatic chain, wherein up to two methylene units of the     aliphatic chain are optionally replaced with —O—, —NR^(a)—, —C(O)—,     or —S(O)_(z); -   L¹ is H; a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from selected from the group consisting of     oxygen, nitrogen, and sulfur; or a C₁₋₆aliphatic chain, wherein up     to two methylene units of the aliphatic chain are optionally     replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); each L¹ is     optionally substituted with C₁₋₄aliphatic, —CN, halo, —OH, or a 3-6     membered non-aromatic ring having 0-2 heteroatoms selected from the     group consisting oxygen, nitrogen, and sulfur; -   L² is H; a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting oxygen, nitrogen, and     sulfur; or a C₁₋₆aliphatic chain, wherein up to two methylene units     of the aliphatic chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z); each L² is optionally substituted with     C₁₋₄aliphatic, —CN, halo, —OH, or a 3-6 membered non-aromatic ring     having 0-2 heteroatoms selected from the group consisting oxygen,     nitrogen, and sulfur; or -   L¹ and L², together with the nitrogen to which they are attached,     form a Ring D; Ring D is optionally substituted with 0-5 occurrences     of J^(G); -   L³ is H, C₁₋₃aliphatic, or CN; -   Ring D is independently a 3-7 membered heterocyclyl ring having 1-2     heteroatoms selected from the group consisting oxygen, nitrogen, and     sulfur; or an 7-12 membered fully saturated or partially unsaturated     bicyclic ring having 1-5 heteroatoms selected from the group     consisting oxygen, nitrogen, and sulfur; -   J^(G) is independently halo, —CN, —N(R^(o))₂;     O; a 3-6 membered carbocyclyl, a 3-6 membered heterocyclyl having     1-2 heteroatoms selected from the group consisting oxygen nitrogen,     and sulfur, or a C₁₋₄alkyl chain, wherein up to two methylene units     of the alkyl chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z); each J^(G) is optionally substituted with 0-2     occurrences of J^(K). -   two occurrences of J^(G) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or -   two occurrences of J^(G), together with Ring D, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   J^(K) is a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; -   z is 0, 1, or 2; and -   R^(a) and R^(o) independently are H or C₁₋₄alkyl.

In some embodiments, R¹ and R³ are fluoro.

In some embodiments, the compound that inhibits ATR is a compound represented by Formula A-4:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound that inhibits ATR is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, a method of treating a proliferative disorder (e.g., cancer) in a subject comprises administering to the subject in need thereof a first dose of an antimetabolite and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase; and administering to the subject in need thereof a second dose of the antimetabolite and between about 12-48 hours later administering to the subject the compound that inhibits ATR protein kinase, wherein the second dose of the antimetabolite is administered between about 6-9 days after the first dose. In some such embodiments, the antimetabolite is selected from the group consisting of Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine, 6-Mercaptopurine, 5-Fluorouracil, Azacitidine and Hydroxyurea. For example, the antimetabolite may be Gemcitabine.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof carboplatin and between about 12-24 hours later administering to the subject a compound that inhibits ATR protein kinase.

In some embodiments, a method for achieving complete response in a subject having colorectal cancer comprises administering to the subject in need thereof a compound that inhibits ATR protein kinase as a monotherapy, wherein the colorectal cancer comprises cells having a defect in ATM.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof carboplatin and between about 12-48 hours later administering to the subject a compound represented by Formula A-2:

or a pharmaceutically acceptable salt thereof, wherein the target AUC of carboplatin is 4 mg/mL·min or 5 mg/mL·min and wherein the dosage of a compound of Formula A-2 is 120 mg/m².

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof carboplatin and between about 12-48 hours later administering to the subject a compound represented by Formula A-2:

or a pharmaceutically acceptable salt thereof, wherein the target AUC of carboplatin is 5 mg/mL·min and wherein the dosage of a compound of Formula A-2 is 90 mg/m².

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof a platinating agent on day 1 and between about 12-48 hours later administering to the subject a first dose of a compound that inhibits ATR protein kinase; and administering to the subject in need thereof a second dose of the compound that inhibits ATR protein kinase on day 9.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof a DNA damaging agent and between about 12-24 hours later administering to the subject a first dose of a compound that inhibits ATR protein kinase; and administering to the subject in need thereof a second dose of the compound that inhibits ATR protein kinase between about 6 to about 9 days after administering the first dose of the compound.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof a platinating agent and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase, wherein the subject is refractory to a treatment with the platinating agent.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof a platinating agent and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase, wherein the subject is resistant to a treatment with the platinating agent.

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof carboplatin and a compound of Formula A-2, wherein the target AUC of carboplatin is between about 3 mg/mL·min and about 6 mg/mL·min and wherein the dosage of a compound of Formula A-2 is between about 60 mg/m² and about 240 mg/m².

In some embodiments, a method of treating a proliferative disorder in a subject comprises administering to the subject in need thereof a compound of Formula A-2, wherein the dosage of a compound of Formula A-2 is between about 120 mg/m² and about 480 mg/m².

In some embodiments, the compound that inhibits ATR is administered about 18-42 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-40 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 12-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 18-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-28 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 24 hours after administration of the DNA damaging agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the optimization of Compound A-2 dose schedule in vitro. PSN1 cancer cells were treated with gemcitabine for 24 hours starting at 0 hours and with Compound A-2 for 2 hours starting at 1, 17, 27, 41, 51 and 65° hours. Cell viability was measured by MTS assay at 96 hours and the data subjected to a statistical Bliss analysis using MacSynergy II software to quantitate synergy as a log volume.

FIG. 2 shows the optimization of intravenous Compound A-2 dose schedule in combination with gemcitabine in vivo. Nude mice bearing PSN1 human pancreatic cancer xenografts were dosed with Compound A-2 or gemcitabine alone or in combination on different schedules, and tumor volumes were monitored for 49 days.

FIG. 3 shows the optimization of intravenous Compound A-2 dose schedule in combination with cisplatin in vivo. SCID mice bearing OD26749 primary human non-small cell lung cancer xenografts were dosed with Compound A-2 or cisplatin alone or in combination on different schedules, and tumor volumes were monitored for 40 days.

FIG. 4 shows radiographs of the left common iliac lymph node before treatment (left) and after 15 months of treatment (right) with 60 mg/m² of Compound A-2 weekly monotherapy.

FIG. 5A shows a graph of plasma concentration versus time for Compound A-2 monotherapy.

FIG. 5B shows a graph of plasma concentration versus time for combination therapy with carboplatin and Compound A-2.

FIG. 6 shows a graph of percentage of pChk1 in the nuclei of cancer cells/mm² tumor before exposure to Compound A-2 (pre-dose) and after exposure to Compound A-2 (post-dose) for various subjects.

FIG. 7 shows radiographs of left peritoneal disease before treatment (left) and after 5 months of treatment (right) after combination treatment with a target AUC of 5 mg/mL·min of carboplatin on day 1 and 90 mg/m² of Compound A-2 on day 2, 24 hours after treatment with carboplatin, and 90 mg/m² of Compound A-2 on day 9.

FIG. 8 shows a tumor response showing changes from baseline in cancer subjects who received treatment of Compound A-2 and cisplatin.

FIG. 9 shows duration of progression-free survival (PFS) from start of treatments of Compound A-2 and cisplatin in subjects having cancer.

FIG. 10 shows radiographs of ovarian cancer before treatment (top) and after 4 cycles of treatment (bottom) with cisplatin and Compound A-2.

FIG. 11 shows radiographs of breast cancer before treatment (top) and after 4 cycles of treatment (bottom) with cisplatin and Compound A-2.

FIG. 12 shows tumor response showing changes from baseline in cancer subjects who received treatment of Compound A-2 as a monotherapy.

FIG. 13 shows duration of progression-free survival (PFS) from start of treatments of Compound A-2 as a monotherapy in subjects having cancer.

FIG. 14 shows tumor response showing changes from baseline in cancer subjects who received treatment of Compound A-2 and carboplatin.

FIG. 15 shows duration of progression-free survival (PFS) from start of treatments of Compound A-2 and carboplatin in subjects having cancer.

FIG. 16 shows tumor response showing changes from baseline in cancer subjects who received treatment of Compound A-2 and gemcitabine, and treatment of Compound A-2, gemcitabine, and cisplatin.

FIG. 17 shows duration of progression-free survival (PFS) from start of treatments of Compound A-2 and gemcitabine in subjects having cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the unexpected discovery that ATR inhibitors administered about 12-48 hours after DNA damaging agents are particularly effective at treating proliferative diseases, such as cancer. As demonstrated in the Examples described below, it has been found that a compound of Formula A-2 (Compound A-2), an ATR inhibitor, synergized with gemcitabine and this synergistic effect markedly increased as Compound A-2 was administered progressively later through the 24 hour gemcitabine dosing period. Synergy was maximal when Compound A-2 was administered about 24 hours after starting gemcitabine treatment; later administration of Compound A-2 was less effective. No synergy was seen when Compound A-2 was administered 48 hours or later, after gemcitabine treatment was started. The strong schedule dependence is attributed to an accumulation of cells in S phase, and concomitant increase in ATR activity that occurs in response to gemcitabine treatment alone. Thus, maximal impact of Compound A-2 is expected at a time when most cells are in S phase as a result of gemcitabine treatment. Extended intervals (>48 hours) between gemcitabine therapy and Compound A-2 exposure allows DNA damage to be repaired, permitting cells to exit S phase and dramatically reducing the impact of ATR inhibition.

Without being bound by theory, it is believed that exposure of cancer cells to certain DNA-damaging agent results in sufficient DNA damage to trigger the DNA damage response and temporary S phase arrest to allow for DNA repair. The DNA damage response is believed to be regulated by two homologous protein kinases, ataxia telangiectasia (ATM) and ataxia telangiectasia Rad3-related (ATR). ATR signals to regulate DNA replication, cell cycle transitions, and DNA repair through the phosphorylation of hundreds of substrates, including checkpoint kinase 1 (Chk1). ATR inhibition during the S phase can thus block effectively the DNA damage repair in cancer cells. It is believed that offsetting treatment with a DNA damaging agent and an ATR inhibitor allows for accumulation of cells in the S phase and the concomitant increase in ATR activity due to the DNA damage response. However, a relatively long offset allows for DNA damage repair, limiting the efficacy of the ATR inhibitor. Accordingly, it is believed that effective treatment of certain cancers may be achieved by administering an ATR inhibitor between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours, about 24 hours, or 24 hours±2 hours) after administration of a DNA-damaging agent. Moreover, administering the ATR inhibitor within the window of highest efficacy can allow efficacy and possible toxicity to be efficiently balanced.

Accordingly, aspects of the disclosure provide a method of treating a proliferative disorder in a subject, the method comprising administering to a subject in need thereof a DNA damaging agent and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase. In some embodiments, the compound that inhibits ATR is administered about 18-42 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-40 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 12-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 18-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-28 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 24 hours, or 24 hours±2 hours, after administration of the DNA damaging agent. In some embodiments, said DNA-damaging agent is chemotherapy or radiation treatment.

In some embodiments in which the DNA damaging agent is given once per treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle), the ATR inhibitor may be administered at least about 12 hours (e.g., at least about 24 hours) after the DNA damaging agent, and optionally a second dose of the ATR inhibitor may be administered at least about 5 days (e.g., at least about 6 days) after a prior (e.g., the immediately prior) administration of the ATR inhibitor. In certain embodiments in which the DNA damaging agent is given once per treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle), the ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after the DNA damaging agent, and optionally a second dose of the ATR inhibitor may be administered between about 5 days to about 9 days after a prior (e.g., the immediately prior) administration of the ATR inhibitor. For instance, in some embodiments, a DNA-damaging agent (e.g., platinating agent) may be administered on day 1 and an ATR inhibitor (e.g., a compound of Formula A-2) may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) later. In some such embodiments, a second dose of the ATR inhibitor (e.g., a compound of Formula A-2) may be administered between about 5 days to about 9 days after a prior (e.g., the immediately prior) administration of the ATR inhibitor.

For instance, the second dose of the ATR inhibitor may be administered after about between about 5 days and about 9 days, between about 5 days and about 8 days, between about 5 days and about 7 days, between about 6 days and about 9 days, between about 6 days and about 8 days, or between about 6 days and about 7 days. In some instances, the second dose of the ATR inhibitor may be administered after between about 6 days and about 8 days or after about 7 days. In one example, a method of treating a proliferative disorder may comprise administering a platinating agent (e.g., carboplatin, cisplatin) on day 1, a first dose of an ATR inhibitor on day 2 (e.g., a compound of Formula A-2) between about 20 hours and about 28 hours (e.g., 24 hours or 24 hours±2 hours) after administration of the platinating agent, and a second dose of the ATR inhibitor about 6 days and about 8 days (e.g., on day 9) after administration of the ATR inhibitor. The method may be part of a 3 week or 4 week treatment cycle. In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the ATR inhibitor for the remaining portion of the treatment cycle. For instance, a method of treating a proliferative disorder using a three week treatment cycle may comprise administering a platinating agent (e.g., carboplatin, cisplatin) on day 1, a first dose of an ATR inhibitor (e.g., a compound of Formula A-2) on day 2 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the platinating agent) and a second dose of the ATR inhibitor on day 9. In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the ATR inhibitor for the remaining portion of the treatment cycle. In certain embodiments, a method of treating a proliferative disorder using a four week treatment cycle may comprise administering a platinating agent (e.g., carboplatin, cisplatin) on day 1, a first dose of an ATR inhibitor (e.g., a compound of Formula A-2) on day 2 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the platinating agent) and a second dose of the ATR inhibitor on day 9. In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the ATR inhibitor for the remaining portion of the treatment cycle.

In some embodiments in which the DNA damaging agent is given twice per treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle), the ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after one administration of the DNA damaging agent or after each administration. In certain embodiments, a first dose of a DNA-damaging agent (e.g., antimetabolite) may be administered on day 1 and an ATR inhibitor (e.g., a compound of Formula A-2) may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) later. In some such embodiments, a second dose of the DNA-damaging agent (e.g., antimetabolite) may be administered between about 5 days to about 9 days after a prior (e.g., immediately prior) administration of the DNA damaging agent. For example, the second dose of the DNA-damaging agent (e.g., antimetabolite) may be administered about between about 5 days and about 9 days, between about 5 days and about 8 days, between about 5 days and about 7 days, between about 6 days and about 9 days, between about 6 days and about 8 days, or between about 6 days and about 7 days after the first dose of the DNA damaging agent. In some instances, the second dose of the DNA-damaging agent may be administered after between about 6 days and about 8 days or after about 7 days. In some embodiments, a second dose of an ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after the second dose of the DNA damaging agent.

In one example, a method of treating a proliferative disorder may comprise administering a first dose of an antimetabolite (e.g., gemcitabine) on day 1, a first dose of an ATR inhibitor (e.g., a compound of Formula A-2) on day 2 between about 20 hours and about 28 hours (e.g., 24 hours or 24 hours±2 hours) after administration of the antimetabolite, and a second dose of the antimetabolite between about 6 days and about 8 days (e.g., on day 8) after the first dose of the antimetabolite. The method, in some instances, further comprises administering a second dose of an ATR inhibitor (e.g., a compound of Formula A-2) between about 20 hours and about 28 hours (e.g., 24 hours or 24 hours±2 hours) after administration of the second dose of the antimetabolite. The method may be part of a 3 week or 4 week treatment cycle. In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the antimetabolite or, when present, the second dose of the ATR inhibitor for the remaining portion of the treatment cycle. For instance, a method of treating a proliferative disorder using a 3 week treatment cycle may comprise administering a first dose of an antimetabolite (e.g., gemcitabine) on day 1, a first dose of an ATR inhibitor on day 2 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the antimetabolite on day 1), and a second dose of the antimetabolite on day 8. The method, in some instances, further comprises administering a second dose of an ATR inhibitor (e.g., a compound of Formula A-2) on day 9 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the second dose of the antimetabolite). In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the antimetabolite or, when present, the second dose of the ATR inhibitor for the remaining portion of the treatment cycle. In certain embodiments, a method of treating a proliferative disorder using a 4 week treatment cycle may comprise administering a first dose of an antimetabolite (e.g., gemcitabine) on day 1, a first dose of an ATR inhibitor on day 2 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the antimetabolite on day 1), and a second dose of the antimetabolite on day 8. The method, in some instances, further comprises administering a second dose of an ATR inhibitor (e.g., a compound of Formula A-2) on day 9 (e.g., about 24 hours, or 24 hours±2 hours, after administration of the second dose of the antimetabolite). In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the antimetabolite or, when present, the second dose of the ATR inhibitor for the remaining portion of the treatment cycle.

In some embodiments in which the DNA damaging agent (e.g., Topo I inhibitor, Topo II inhibitor) is administered three or more times per treatment cycle (e.g., 3-5 administrations), the ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after at least one administration of the DNA damaging agent (e.g., after one administration, after each of two administrations, after each of three administrations) or after each administration.

In some embodiments, two or more different DNA damaging agents may be administered within a treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle). The DNA damaging agents may differ in mechanism of action and/or administration frequency. For instance, a first DNA-damaging agent (e.g., antimetabolite) administered twice per treatment cycle and a second DNA damaging agent (e.g., platinating agent) administered once per treatment cycle may be used. In some such embodiments, the first DNA-damaging agent and a second DNA damaging agent may be administered as described above with respect to the administration of a single DNA-damaging agent. The ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after at least one DNA damaging agent (e.g., two DNA damaging agents, after each of two administrations of a DNA damaging agent.

In one example, a platinating agent and an antimetabolite (e.g., carboplatin and gemcitabine, cisplatin and gemcitabine) may be administered on day 1 and a first dose of an ATR inhibitor (e.g., a compound of Formula A-2) may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after the platinating agent and the antimetabolite. In some such embodiments, a second dose of the antimetabolite may be administered between about 6 days and about 8 days (e.g., on day 8) after the first dose of the antimetabolite. The method, in some instances, further comprises administering a second dose of an ATR inhibitor (e.g., a compound of Formula A-2) between about 20 hours and about 28 hours (e.g., 24 hours or 24 hours±2 hours) after administration of the second dose of the antimetabolite. The method may be part of a 3 week or 4 week treatment cycle. In some such embodiments, a DNA-damaging agent or ATR inhibitor may not be administered after the second dose of the antimetabolite or, when present, the second dose of the ATR inhibitor for the remaining portion of the treatment cycle. In other embodiments, a third dose of the ATR inhibitor may be administered between about 6 and about 8 days after the second dose of the ATR inhibitor.

In some embodiments, a DNA damaging agent, ATR inhibitor, and an additional therapeutic agent may be administered within a treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle). In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, such as a taxane (e.g., taxol, docetaxol, cabazitaxel). For instance, a platinating agent (e.g., carboplatin, cisplatin), ATR inhibitor (e.g., a compound of Formula A-2), and taxol may be administered within a single treatment cycle (e.g., 3 week treatment cycle, 4 week treatment cycle). In some such embodiments, the DNA-damaging agent and ATR inhibitor may be administered as described herein. For instance, the ATR inhibitor may be administered between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after a DNA damaging agent.

As used herein, the term “treatment cycle” has its ordinary meaning in the art and may refer to a course of treatment that is repeated on a regular schedule, including periods of rest. For example, a treatment cycle of four weeks may include administration of agents during week one followed by three weeks of rest (e.g., no treatment). In general, an ATR inhibitor may be administered at least once per treatment cycle and between about 12 hours and about 48 hours (e.g., between about 12 hours and about 36 hours, between about 20 hours and about 28 hours) after a DNA damaging agent. In some embodiments, the methods, described herein, may be part of a 3 week or 4 week treatment cycle.

In some embodiments, treatment of a proliferative disorder using the methods described herein may result in a RECIST stable disease, a RECIST partial response, or a RECIST complete response. For instance, treatment may result in a RECIST partial or a RECIST complete response. As used herein, the term “RECIST partial response” has its ordinary meaning in the art and may refer to a 30% decrease in the sum of the longest diameter of target lesions as determined according to the RECIST (i.e., Response Evaluation Criteria in Solid Tumors) guidelines version 1.1 (see Eisenhauer et. al., Eur. J. Cancer. 45 (2009) 228-247). As used herein, the term “RECIST complete response” has its ordinary meaning in the art and may refer to the disappearance of all target lesions as determined according to the RECIST guidelines version 1.1. As used herein, the term “RECIST progressive disease” has its ordinary meaning in the art and may refer to a 20% increase in the sum of the longest diameter of target lesions as determined according to the RECIST guidelines version 1.1. As used herein, the term “RECIST stable disease” has its ordinary meaning in the art and may refer to small changes that do not meet above criteria as determined according to the RECIST guidelines version 1.1.

In general, treatment of a proliferative disorder (e.g., cancer) with the methods described herein may reverse, alleviate, delaying the onset of, or inhibit the progress of the proliferative disorder. In some embodiments, the methods described herein may decrease the sum of the longest diameter of target lesions, decrease the sum of the longest diameter of non-target lesions, and/or decrease tumor burden by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%. In certain embodiments, the methods described herein may decrease the sum of the longest diameter of target lesions, decrease the sum of the longest diameter of non-target lesions, and/or decrease tumor burden by between about 20% and about 60% or between about 40% and about 60%.

In some embodiments, the methods described herein may be particularly advantageous for the treatment of proliferative disorders in subjects that are refractory, resistant, or sensitive to one or more DNA damaging. In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer, colorectal cancer, breast cancer) in a subject that is refractory to a platinating agent (e.g., cisplatin, carboplatin). In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer, breast cancer) in a subject that is refractory to an antimetabolite (e.g., gemcitabine). For example, as described in more detail in the Examples, it was surprisingly found that treatment of a human subject having metastatic high grade serous ovarian cancer having gBRCA1 and TP53 mutations with peritoneal, liver and nodal disease that was refractory to carboplatin and gemcitabine had a RECIST partial response after treatment with carboplatin and a compound of Formula A-2 as described herein.

In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer, colorectal cancer, breast cancer) in a subject that is resistant to a platinating agent (e.g., cisplatin, carboplatin). In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer) in a subject that is resistant to an antimetabolite (e.g., gemcitabine). In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer, breast cancer, colorectal cancer) in a subject that is sensitive to a platinating agent (e.g., cisplatin, carboplatin). In certain embodiments, the methods described herein may be used to treat a proliferative disorder (e.g., ovarian cancer, lung cancer breast cancer, colorectal cancer) in a subject that is sensitive to an antimetabolite (e.g., gemcitabine). For example, as described in more detail in the Examples, it was surprisingly found that treatment of a human subject, who was a BRCA2 W2626Q (8106 C>G) carrier, having CA 125 positive ovarian cancer that was resistant to carboplatin had a RECIST partial response after treatment with cisplatin and a compound of Formula A-2 as described herein.

As used herein, the terms “refractory” has its ordinary meaning in the art and may refer to a proliferative disorder that progresses during treatment with an agent (e.g., DNA damaging agent) (first line treatment). As used herein, the terms “resistant” has its ordinary meaning in the art and may refer to a proliferative disorder that recurs within a certain period of time after completing treatment with an agent (e.g., DNA damaging agent). As used herein, the terms “sensitive” has its ordinary meaning in the art and may refer to a proliferative disorder that recurs after a certain period of time from completing treatment with an agent (e.g., DNA damaging agent). In general, recurrence occurs after a longer period of time for a sensitive cancer than for a resistant cancer. The periods of time to classify a proliferative disorder as resistance or sensitive would be known to those of ordinary skill in the art and may depend on certain factors, such as the type of cancer, the treatment used, and the stage of cancer, amongst others. For instance, resistant ovarian cancer may refer to ovarian cancer that recurs within 6 months from completing treatment. Sensitive ovarian cancer may refer to ovarian cancer that recurs after greater than 6 months from completing treatment. For instance, resistant small cell lung cancer (SCLC) may refer to SCLC that recurs within 3 months from completing treatment. Sensitive SCLC may refer to SCLC that recurs after greater than 3 months from completing treatment.

In some embodiments, the methods described herein may be particularly advantageous for the treatment of proliferative disorders having a defect in the ATM signaling cascade. In some embodiments, the defect is altered expression or activity of one or more of the following: ATM and p53. In certain embodiments, the proliferative disorder may have a mutation (e.g., somatic) in p53. For example, treatment of a proliferative disorder (e.g., ovarian cancer) having a somatic mutation in the TP53 gene by administering a platinating agent (e.g., carboplatin, cisplatin) on day 1, a first dose of an ATR inhibitor (e.g., a compound of Formula A-2) between about 20 hours and about 28 hours (e.g., 24 hours or 24 hours±2 hours) after administration of the platinating agent on day 1, and a second dose of the ATR inhibitor between about 6 days and about 8 days (e.g., on day 9) after the first dose, as part of a three or four week treatment cycle including a rest period after the second dose of the ATR inhibitor, may result in at least a RECIST partial response. In some such embodiments, the methods described herein may decrease the sum of the longest diameter of target lesions, decrease the sum of the longest diameter of non-target lesions, and/or decrease tumor burden by between about 20% and about 60% or between about 40% and about 60%.

In some embodiments, the proliferative disorder may have a complete loss of ATM signaling. For example, treatment of a proliferative disorder (e.g., colorectal cancer) having a complete loss of ATM signaling with a monotherapy (e.g., at a dosage of about 60 mg/m², between about 60 mg/m² and about 480 mg/m², about 120 mg/m², about 240 mg/m², about 480 mg/m²) or a combination therapy with a DNA damaging agent, as described herein may result in a decrease in the sum of the longest diameter of target lesions, decrease in the sum of the longest diameter of non-target lesions, and/or decrease in tumor burden by at least about 80% or RECIST complete response.

Compounds

In some aspects of the present disclosure, the compound that inhibits ATR protein kinase is a compound represented by Formula A-I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-4     heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, wherein said monocyclic aryl or     heteroaryl ring is optionally fused to another ring to form an 8-10     membered bicyclic aryl or heteroaryl ring having 0-6 heteroatoms     independently selected from the group consisting of nitrogen,     oxygen, and sulfur; each R¹ is optionally substituted with 1-5 J¹     groups; -   R² is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-3     heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, wherein said monocyclic aryl or     heteroaryl ring is optionally fused to another ring to form an 8-10     membered bicyclic aryl or heteroaryl ring having 0-4 heteroatoms     independently selected from the group consisting of nitrogen,     oxygen, and sulfur; each R² is optionally substituted with 1-5 J²     groups; -   L is —C(O)NH— or —C(O)N(C₁₋₆alkyl)-; -   n is 0 or 1; -   each J¹ and J² is independently halo, —CN, —NO₂, —V¹—R, or     —(V²)_(m)-Q; -   V¹ is a C₁₋₁₀aliphatic chain, wherein 0-3 methylene units are     optionally and independently replaced with O, NR″, S, C(O), S(O), or     S(O)₂; V¹ is optionally substituted with 1-6 occurrences of J^(V1); -   V² is a C₁₋₁₀aliphatic chain, wherein 0-3 methylene units are     optionally and independently replaced with 0, NR″, S, C(O), S(O), or     S(O)₂; V² is optionally substituted with 1-6 occurrences of J^(V2); -   m is 0 or 1; -   Q is a 3-8 membered saturated or unsaturated monocyclic ring having     0-4 heteroatoms independently selected from the group consisting of     nitrogen, oxygen, and sulfur, or a 9-10 membered saturated or     unsaturated bicyclic ring having 0-6 heteroatoms independently     selected from the group consisting of nitrogen, oxygen, and sulfur;     each Q is optionally substituted with 0-5 J^(Q); -   each J^(V1) or J^(V2) is independently halogen, CN, NH₂, NO₂,     C₁₋₄aliphatic, NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, OH,     O(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,     C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, NHCO(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)CO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic),     NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein     said C₁₋₄aliphatic is optionally substituted with halo; -   R is H or C₁₋₆aliphatic, wherein said C₁₋₆aliphatic is optionally     substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),     NO₂, CN, CO₂H, CO₂(C₁₋₄aliphatic), CO(C₁₋₄aliphatic),     O(haloC₁₋₄aliphatic), or haloC₁₋₄aliphatic; -   each J^(Q) is independently halo, oxo, CN, NO₂, X—R, or —(X)_(p)-Q⁴; -   p is 0 or 1; -   X is C₁₋₁₀aliphatic, wherein 1-3 methylene units of said     C₁₋₆aliphatic are optionally replaced with —NR, —O—, —S—, C(O),     S(O)₂, or S(O); wherein X is optionally and independently     substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),     N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),     NO₂, CN, CO(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,     C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, SO(C₁₋₄aliphatic),     SO₂(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic), SO₂N(C₁₋₄aliphatic)₂,     NHC(O)(C₁₋₄aliphatic), N(C₁₋₄aliphatic)C(O)(C₁₋₄aliphatic), wherein     said C₁₋₄aliphatic is optionally substituted with 1-3 occurrences of     halo; Q⁴ is a 3-8 membered saturated or unsaturated monocyclic ring     having 0-4 heteroatoms independently selected from the group     consisting of nitrogen, oxygen, and sulfur, or a 8-10 membered     saturated or unsaturated bicyclic ring having 0-6 heteroatoms     independently selected from the group consisting of nitrogen,     oxygen, and sulfur; each Q⁴ is optionally substituted with 1-5 JQ⁴; -   J^(Q4) is halo, CN, or C₁₋₄alkyl, wherein up to 2 methylene units     are optionally replaced with 0,

NR*, S, C(O), S(O), or S(O)₂;

-   R is H or C₁₋₄alkyl, wherein said C₁₋₄alkyl is optionally     substituted with 1-4 halo; -   R″ and R* are each independently H, C₁₋₄alkyl, or is absent; wherein     said C₁₋₄alkyl is optionally substituted with 1-4 halo.

In some embodiments, L is —C(O)NH—; and R¹ and R² are phenyl.

In another embodiment the compound that inhibits ATR kinase is a compound represented by Formula A-I-a:

or a pharmaceutically salt thereof, wherein:

-   Ring A is

-   J⁵o is H, F, Cl, C₁₋₄aliphatic, O(C₁₋₃aliphatic), or OH; -   J⁵p is

-   J⁵p₁ is H, C₁₋₄aliphatic, oxetanyl, tetrahydrofuranyl,     tetrahydropyranyl; wherein J⁵p₁ is optionally substituted with 1-2     occurrences of OH or halo; -   J⁵p₂ is H, methyl, ethyl, CH₂F, CF₃, or CH₂OH; -   J²o is H, CN, or SO₂CH₃; -   J²m is H, F, Cl, or methyl; -   J²p is —SO₂(C₁₋₆alkyl), —SO₂(C₃₋₆cycloalkyl), —SO₂(4-6 membered     heterocyclyl), —SO₂(C₁₋₄alkyl)N(C₁₋₄alkyl)₂, or —SO₂(C₁₋₄alkyl)-(4-6     membered heterocyclyl), wherein said heterocyclyl contains 1     heteroatom selected from the group consisting of oxygen, nitrogen,     and sulfur; and wherein said J²p is optionally substituted with 1-3     occurrences halo, OH, or O(C₁₋₄alkyl).

In some embodiments, Ring A is

In other embodiments, Ring A is

In some embodiments, the compound that inhibits ATR kinase is a compound represented by Formula A-1:

or a pharmaceutically acceptable salt thereof, or a compound represented by Formula A-2:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound that inhibits ATR kinase is a compound represented by Formula A-1:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound that inhibits ATR kinase is a compound represented by Formula A-2:

or a pharmaceutically acceptable salt thereof.

In another aspect of the present disclosure, the compound that inhibits ATR protein kinase is represented by Formula A-II:

or a pharmaceutically salt or derivative thereof, wherein:

-   R¹⁰ is selected from fluoro, chloro, or —C(J¹⁰)₂CN; -   J¹⁰ is independently H or C₁₋₂alkyl; or -   two occurrences of J¹⁰, together with the carbon atom to which they     are attached, form a 3-4 membered optionally substituted carbocyclic     ring; -   R²⁰ is H, halo, —CN, NH₂, a C₁₋₂alkyl optionally substituted with     0-3 occurrences of fluoro; or a C₁₋₃aliphatic chain, wherein up to     two methylene units of the aliphatic chain are optionally replaced     with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); -   R³ is H, halo, C₁₋₄alkyl optionally substituted with 1-3 occurrences     of halo, C₃₋₄cycloalkyl, —CN, or a C₁₋₃aliphatic chain, wherein up     to two methylene units of the aliphatic chain are optionally     replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); -   R⁴ is Q¹ or a C₁₋₁₀aliphatic chain, wherein up to four methylene     units of the aliphatic chain are optionally replaced with —O—,     —NR^(a)—, —C(O)—, or —S(O)_(z)—; each R⁴ is optionally substituted     with 0-5 occurrences of J^(Q1); or -   R³ and R⁴, taken together with the atoms to which they are bound,     form a 5-6 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; the ring formed by R³ and R⁴ is optionally substituted     with 0-3 occurrences of J^(Z); -   Q¹ is a 3-7 membered fully saturated, partially unsaturated, or     aromatic monocyclic ring, the 3-7 membered ring having 0-3     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or an 7-12 membered fully saturated, partially     unsaturated, or aromatic bicyclic ring having 0-5 heteroatoms     selected from the group consisting of oxygen, nitrogen, and sulfur; -   J^(Z) is independently C₁₋₆aliphatic, ═O, halo, or     O; -   J^(Q1) is independently-CN, halo, ═O, Q², or a C₁₋₈aliphatic chain,     wherein up to three methylene units of the aliphatic chain are     optionally replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z)—; each     occurrence of J^(Q1) is optionally substituted by 0-3 occurrences of     J^(R); or -   two occurrences of J^(Q1) on the same atom, taken together with the     atom to which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; wherein the ring formed by two occurrences of J^(Q1) is     optionally substituted with 0-3 occurrences of J^(X); or -   two occurrences of J^(Q1), together with Q¹, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   Q² is independently selected from a 3-7 membered fully saturated,     partially unsaturated, or aromatic monocyclic ring having 0-3     heteroatoms selected from oxygen, nitrogen, or sulfur; or an 7-12     membered fully saturated, partially unsaturated, or aromatic     bicyclic ring having 0-5 heteroatoms selected from oxygen, nitrogen,     or sulfur; -   J^(R) is independently —CN, halo, ═O,     O; Q³, or a C₁₋₆aliphatic chain, wherein up to three methylene units     of the aliphatic chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z)—; each J^(R) is optionally substituted with 0-3     occurrences of J^(T); or -   two occurrences of J^(R) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from oxygen, nitrogen, or sulfur; wherein the     ring formed by two occurrences of J^(R) is optionally substituted     with 0-3 occurrences of J^(X); or -   two occurrences of J^(R), together with Q², form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   Q³ is a 3-7 membered fully saturated, partially unsaturated, or     aromatic monocyclic ring having 0-3 heteroatoms selected from the     group consisting of oxygen, nitrogen, and sulfur; or an 7-12     membered fully saturated, partially unsaturated, or aromatic     bicyclic ring having 0-5 heteroatoms selected from the group     consisting of oxygen, nitrogen, and sulfur; -   J^(X) is independently —CN, ═O, halo, or a C₁₋₄aliphatic chain,     wherein up to two methylene units of the aliphatic chain are     optionally replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z)—; -   J^(T) is independently halo, —CN,     O; ═O, —OH, a C₁₋₆aliphatic chain, wherein up to two methylene units     of the aliphatic chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z)—; or a 3-6 membered non-aromatic ring having     0-2 heteroatoms selected from the group consisting of oxygen,     nitrogen, and sulfur; each occurrence of J^(T) is optionally     substituted with 0-3 occurrences of J^(M); or -   two occurrences of J^(T) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or -   two occurrences of J^(T), together with Q³, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   J^(M) is independently halo or C₁₋₆aliphatic; -   z is 0, 1 or 2; and -   R^(a) is independently H or C₁₋₄aliphatic.

In some embodiments, R¹⁰ and R³ are fluoro.

In other embodiments, R⁴ is Q¹.

In still other embodiments, Q¹ is independently piperidinyl and imidazolyl.

In yet another embodiment, the compound that inhibits ATR is a compound represented by Formula A-3:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound that inhibits ATR is represented by Formula A-II-a:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

-   R¹⁰ is fluoro, chloro, or —C(J¹⁰)₂CN; -   J¹⁰ is independently H or C₁₋₂alkyl; or -   two occurrences of J¹, together with the carbon atom to which they     are attached, form an optionally substituted 3-4 membered     carbocyclic ring; -   R³ is H; chloro; fluoro; C₁₋₄alkyl optionally substituted with 1-3     occurrences of halo; C₃₋₄cycloalkyl; —CN; or a C₁₋₃aliphatic chain,     wherein up to two methylene units of the aliphatic chain are     optionally replaced with —O—, —NR^(a)—, —C(O)—, or —S(O)_(z); -   L¹ is H; a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or a C₁₋₆aliphatic chain, wherein up to two methylene     units of the aliphatic chain are optionally replaced with —O—,     —NR^(a)—, —C(O)—, or —S(O)_(z); each L¹ is optionally substituted     with C₁₋₄aliphatic; —CN; halo; —OH; or a 3-6 membered non-aromatic     ring having 0-2 heteroatoms selected from the group consisting of     oxygen, nitrogen, and sulfur; -   L² is H; a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or a C₁₋₆aliphatic chain, wherein up to two methylene     units of the aliphatic chain are optionally replaced with —O—,     —NR^(a)—, —C(O)—, or —S(O)_(z); each L² is optionally substituted     with C₁₋₄aliphatic; —CN; halo; —OH; or a 3-6 membered non-aromatic     ring having 0-2 heteroatoms selected from the group consisting of     oxygen, nitrogen, and sulfur; or -   L¹ and L², together with the nitrogen to which they are attached,     form a Ring D; Ring D is optionally substituted with 0-5 occurrences     of J^(G); -   L³ is H, C₁₋₃aliphatic, or CN; -   Ring D is a 3-7 membered heterocyclyl ring having 1-2 heteroatoms     selected from the group consisting of oxygen, nitrogen, and sulfur;     or an 7-12 membered fully saturated or partially unsaturated     bicyclic ring having 1-5 heteroatoms selected from the group     consisting of oxygen, nitrogen, and sulfur; -   J^(G) is independently halo; —CN; —N(R^(o))₂;     O; a 3-6 membered carbocycyl; a 3-6 membered heterocyclyl having 1-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or a C₁₋₄alkyl chain, wherein up to two methylene units     of the alkyl chain are optionally replaced with —O—, —NR^(a)—,     —C(O)—, or —S(O)_(z); each J^(G) is optionally substituted with 0-2     occurrences of J^(K). -   two occurrences of J^(G) on the same atom, together with the atom to     which they are joined, form a 3-6 membered ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; or -   two occurrences of J^(G), together with Ring D, form a 6-10 membered     saturated or partially unsaturated bridged ring system; -   J^(K) is a 3-7 membered aromatic or non-aromatic ring having 0-2     heteroatoms selected from the group consisting of oxygen, nitrogen,     and sulfur; -   z is 0, 1, or 2; and -   R^(a) and R^(o) are independently H or C₁₋₄alkyl.

In another embodiment, R¹ and R³ are fluoro.

In still other embodiments, the compound that inhibits ATR is a compound represented by Formula A-4:

or a pharmaceutically acceptable salt thereof.

In yet another embodiment, the compound is an ATR inhibitor selected from a compound described in WO 2013/049726, WO 2013/152298, WO 2013/049859, US-2013-0089625, US-2013-0115312, US-2014-0107093, US-2013-0096139, WO 2011/143426, US-2013-0095193, WO 2014/055756, WO 2011/143419, WO 2011/143422, WO 2011/143425, US-2013-0115311, US-2013-0115312, US-2013-0115313, US-2013-0115314, WO 2011/163527, WO 2012/178123, WO 2012/178124, WO 2012/178125, US-2014-0113005, WO2013/049726, WO 2013/071085, WO 2010/071837, WO 2014/089379, WO 2014/143242, WO 2014/143241, WO 2015/084384, and/or WO 2014/143240. In certain embodiments, the ATR inhibitor is a compound of Formula (A-I) or (A-II). In certain embodiments, the ATR inhibitor is a compound of Formula A-1, A-2, A-3, or A-4.

In yet another embodiment, the compound is an ATR inhibitor selected from a compound described in WO 2015/187451, WO 2015/085132, WO 2014/062604; WO 2014/143240; WO 2013/071094; WO 2013/071093; WO 2013/071090; WO 2013/071088; WO 2013/049859; WO 2013/049719; WO 2013/049720; WO 2013/049722; WO 2012/138,938; WO 2011/163527; WO 2011/143,423; WO 2011/143,426; WO 2011/143,399; and/or WO 2010/054398.

In certain embodiments, the compound that inhibits ATR is selected from a compound described in WO 2013/014448. In certain embodiments, the compound that inhibits ATR is AZD-6738.

For purposes of this application, it will be understood that the terms embodiment, example, and aspect are used interchangeably.

For example, for the purposes of this application, it will be understood that when two occurrences of J^(Q1), together with Q¹, form a bridged ring system, the two occurrences of J^(Q1) are attached to separate atoms of Q¹. Additionally, when two occurrences of J^(R), together with Q², form a bridged ring system, the two occurrences of J^(R) are attached to separate atoms of Q². Moreover, when two occurrences of J^(T), together with Q³, form a bridged ring system, the two occurrence of J^(T) are attached to separate atoms of Q³. Finally, when two occurrences of J^(G), together with Ring D, form a bridged ring system, the two occurrences of J^(G) are attached to separate atoms of Ring D.

For purposes of this application, it will be understood that the terms ATR, ATR kinase, and ATR protein kinase, as well as an ATR inhibitor and a compound that inhibits ATR, are used interchangeably.

It will be understood by those skilled in the art that the arrow in

O represents a dative bond.

This application refers to various issued patent, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference.

Compounds of this invention include those described generally herein, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

As described herein, a specified number range of atoms includes any integer therein. For example, a group having from 1-4 atoms could have 1, 2, 3, or 4 atoms.

As described herein, compounds of the invention may optionally be substituted with one or more substituents, such as are illustrated generally herein, or as exemplified by particular classes, subclasses, and species of the invention. It will be appreciated that the phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” In general, the term “substituted”, whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.

Unless otherwise indicated, a substituent connected by a bond drawn from the center of a ring means that the substituent can be bonded to any position in the ring. In example i below, for instance, J¹ can be bonded to any position on the pyridyl ring. For bicyclic rings, a bond drawn through both rings indicates that the substituent can be bonded from any position of the bicyclic ring. In example ii below, for instance, J¹ can be bonded to the 5-membered ring (on the nitrogen atom, for instance), and to the 6-membered ring.

The term “stable”, as used herein, refers to compounds that are not substantially altered when patiented to conditions to allow for their production, detection, recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

The term “dative bond”, as used herein, is defined as the coordination bond formed upon interaction between molecular species, one of which serves as a donor and the other as an acceptor of the electron pair to be shared in the complex formed.

The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched), branched, or cyclic, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation that has a single point of attachment to the rest of the molecule.

Unless otherwise specified, aliphatic groups contain 1-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-10 aliphatic carbon atoms. In other embodiments, aliphatic groups contain 1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms, and in yet other embodiments aliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groups may be linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups. Specific examples include, but are not limited to, methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl, ethynyl, and tert-butyl. Aliphatic groups may also be cyclic, or have a combination of linear or branched and cyclic groups. Examples of such types of aliphatic groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, —CH₂— cyclopropyl, CH₂CH₂CH(CH₃)-cyclohexyl.

The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl”) refers to a monocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members. Examples of cycloaliphatic groups include, but are not limited to, cycloalkyl and cycloalkenyl groups. Specific examples include, but are not limited to, cyclohexyl, cyclopropenyl, and cyclobutyl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used herein means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members are an independently selected heteroatom. In some embodiments, the “heterocycle”, “heterocyclyl”, or “heterocyclic” group has three to fourteen ring members in which one or more ring members is a heteroatom independently selected from oxygen, sulfur, nitrogen, or phosphorus, and each ring in the system contains 3 to 7 ring members.

Examples of heterocycles include, but are not limited to, 3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl, 2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl, 4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, 5-imidazolidinyl, indolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and 1,3-dihydro-imidazol-2-one.

Cyclic groups, (e.g. cycloaliphatic and heterocycles), can be linearly fused, bridged, or spirocyclic.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation. As would be known by one of skill in the art, unsaturated groups can be partially unsaturated or fully unsaturated. Examples of partially unsaturated groups include, but are not limited to, butene, cyclohexene, and tetrahydropyridine. Fully unsaturated groups can be aromatic, anti-aromatic, or non-aromatic. Examples of fully unsaturated groups include, but are not limited to, phenyl, cyclooctatetraene, pyridyl, thienyl, and 1-methylpyridin-2(1H)-one.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkyl group, as previously defined, attached through an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom.

The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy” mean alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. This term includes perfluorinated alkyl groups, such as —CF₃ and —CF₂CF₃.

The terms “halogen”, “halo”, and “hal” mean F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the term “aryl ring”.

The term “heteroaryl”, used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in the system contains 3 to 7 ring members. The term “heteroaryl” may be used interchangeably with the term “heteroaryl ring” or the term “heteroaromatic”. Examples of heteroaryl rings include, but are not limited to, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl, 1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl).

It shall be understood that the term “heteroaryl” includes certain types of heteroaryl rings that exist in equilibrium between two different forms. More specifically, for example, species such hydropyridine and pyridinone (and likewise hydroxypyrimidine and pyrimidinone) are meant to be encompassed within the definition of “heteroaryl.”

The term “protecting group” and “protective group” as used herein, are interchangeable and refer to an agent used to temporarily block one or more desired functional groups in a compound with multiple reactive sites. In certain embodiments, a protecting group has one or more, or preferably all, of the following characteristics: a) is added selectively to a functional group in good yield to give a protected substrate that is b) stable to reactions occurring at one or more of the other reactive sites; and c) is selectively removable in good yield by reagents that do not attack the regenerated, deprotected functional group. As would be understood by one skilled in the art, in some cases, the reagents do not attack other reactive groups in the compound. In other cases, the reagents may also react with other reactive groups in the compound. Examples of protecting groups are detailed in Greene, T. W., Wuts, P. G in “Protective Groups in Organic Synthesis”, Third Edition, John Wiley & Sons, New York: 1999 (and other editions of the book), the entire contents of which are hereby incorporated by reference. The term “nitrogen protecting group”, as used herein, refers to an agent used to temporarily block one or more desired nitrogen reactive sites in a multifunctional compound. Preferred nitrogen protecting groups also possess the characteristics exemplified for a protecting group above, and certain exemplary nitrogen protecting groups are also detailed in Chapter 7 in Greene, T. W., Wuts, P. G in “Protective Groups in Organic Synthesis”, Third Edition, John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference.

In some embodiments, a methylene unit of an alkyl or aliphatic chain is optionally replaced with another atom or group. Examples of such atoms or groups include, but are not limited to, nitrogen, oxygen, sulfur, —C(O)—, —C(═N—CN)—, —C(═NR)—, —C(═NOR)—, —SO—, and —SO₂—. These atoms or groups can be combined to form larger groups. Examples of such larger groups include, but are not limited to, —OC(O)—, —C(O)CO—, —CO₂—, —C(O)NR—, —C(═N—CN), —NRCO—, —NRC(O)O—, —SO₂NR—, —NRSO₂—, —NRC(O)NR—, —OC(O)NR—, and —NRSO₂NR—, wherein R is, for example, H or C₁₋₆aliphatic. It should be understood that these groups can be bonded to the methylene units of the aliphatic chain via single, double, or triple bonds. An example of an optional replacement (nitrogen atom in this case) that is bonded to the aliphatic chain via a double bond would be —CH₂CH═N—CH₃. In some cases, especially on the terminal end, an optional replacement can be bonded to the aliphatic group via a triple bond. One example of this would be CH₂CH₂CH₂C≡N. It should be understood that in this situation, the terminal nitrogen is not bonded to another atom.

It should also be understood that, the term “methylene unit” can also refer to branched or substituted methylene units. For example, in an isopropyl moiety [—CH(CH₃)₂], a nitrogen atom (e.g. NR) replacing the first recited “methylene unit” would result in dimethylamine [—N(CH₃)₂]. In instances such as these, one of skill in the art would understand that the nitrogen atom will not have any additional atoms bonded to it, and the “R” from “NR” would be absent in this case.

Unless otherwise indicated, the optional replacements form a chemically stable compound. Optional replacements can occur both within the chain and/or at either end of the chain; i.e. both at the point of attachment and/or also at the terminal end. Two optional replacements can also be adjacent to each other within a chain so long as it results in a chemically stable compound. For example, a C₃ aliphatic can be optionally replaced by 2 nitrogen atoms to form —C—N≡N. The optional replacements can also completely replace all of the carbon atoms in a chain. For example, a C₃ aliphatic can be optionally replaced by —NR—, —C(O)—, and —NR— to form —NRC(O)NR— (a urea).

Unless otherwise indicated, if the replacement occurs at the terminal end, the replacement atom is bound to a hydrogen atom on the terminal end. For example, if a methylene unit of —CH₂CH₂CH₃ were optionally replaced with —O—, the resulting compound could be —OCH₂CH₃, —CH₂OCH₃, or —CH₂CH₂OH. It should be understood that if the terminal atom does not contain any free valence electrons, then a hydrogen atom is not required at the terminal end (e.g., —CH₂CH₂CH═O or —CH₂CH₂CN).

Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, and rotational) forms of the structure. For example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are included in this invention. As would be understood to one skilled in the art, a substituent can freely rotate around any rotatable bonds. For example, a substituent drawn as

also represents

Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, geometric, conformational, and rotational mixtures of the present compounds are within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

Additionally, unless otherwise indicated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools or probes in biological assays.

The compounds disclosed herein can be prepared by any suitable methods known in the art, for example, WO 2015/187451, WO 2015/085132, WO 2014/062604; WO 2014/143240; WO 2013/071094; WO 2013/071093; WO 2013/071090; WO 2013/071088; WO 2013/049859; WO 2013/049719; WO 2013/049720; WO 2013/049722; WO 2012/138,938; WO 2011/163527; WO 2011/143,423; WO 2011/143,426; WO 2011/143,399; WO 2010/054398; WO 2013/049726, WO 2013/152298, WO 2013/049859, US-2013-0089625, US-2013-0115312, US-2014-0107093, US-2013-0096139, WO 2011/143426, US-2013-0095193, WO 2014/055756, WO 2011/143419, WO 2011/143422, WO 2011/143425, US-2013-0115311, US-2013-0115312, US-2013-0115313, US-2013-0115314, WO 2011/163527, WO 2012/178123, WO 2012/178124, WO 2012/178125, US-2014-0113005, WO2013/049726, WO 2013/071085, WO 2010/071837, WO 2014/089379, WO 2014/143242, WO 2014/143241, WO 2015/084384, and/or WO 2014/143240.

DNA Damaging Agents

In some aspects of the present disclosure, the DNA damaging agent is radiation therapy. In certain embodiments, the DNA-damaging agent comprises chemotherapy. In certain embodiments, the ATR inhibitor is a compound of Formula A-1, a compound of Formula A-2, a compound of Formula A-3, a compound of Formula A-4, or AZD-6738, and the DNA-damaging agent is radiation or chemotherapy.

In certain embodiments, the DNA damaging agent comprises radiation therapy. Examples of radiation therapy include, but are not limited to, ionizing radiation, gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes and radiosensitizers. Radiosensitizers work in various different ways, including, but not limited to, making cancer cells more sensitive to radiation, working in synergy with radiation to provide an improved synergistic effect, acting additively with radiation, or protecting surrounding healthy cells from damage caused by radiation.

In certain embodiments, the DNA-damaging agent comprises chemotherapy. Examples of chemotherapy include, but are not limited to, platinating agents, such as Carboplatin, Oxaliplatin, Cisplatin, Nedaplatin, Satraplatin, Lobaplatin, Triplatin, Tetranitrate, Picoplatin, Prolindac, Aroplatin and other derivatives; topo I inhibitors, such as Camptothecin, Topotecan, irinotecan/SN38, rubitecan, Belotecan, and other derivatives; topo II inhibitors, such as Etoposide (VP-16), Daunorubicin, Doxorubicin, Mitoxantrone, Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Amsacrine, Pirarubicin, Valrubicin, Zorubicin, Teniposide and other derivatives; antimetabolites, such as Folic family (Methotrexate, Pemetrexed, Raltitrexed, Aminopterin, and relatives); Purine antagonists (Thioguanine, Fludarabine, Cladribine, 6-Mercaptopurine, Pentostatin, clofarabine and relatives) and Pyrimidine antagonists (Cytarabine, Floxuridine, Azacitidine, Tegafur, Carmofur, Capacitabine, Gemcitabine, hydroxyurea, 5-Fluorouracil(5FU), and relatives); alkylating agents, such as Nitrogen mustards (e.g., Cyclophosphamide, Melphalan, Chlorambucil, mechlorethamine, Ifosfamide, mechlorethamine, Trofosfamide, Prednimustine, Bendamustine, Uramustine, Estramustine, and relatives); nitrosoureas (e.g., Carmustine, Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin, and relatives); Triazenes (e.g., Dacarbazine, Altretamine, Temozolomide, and relatives); Alkyl sulphonates (e.g., Busulfan, Mannosulfan, Treosulfan, and relatives); Procarbazine; Mitobronitol, and Aziridines (e.g., Carboquone, Triaziquone, ThioTEPA, triethylenemalamine, and relatives); antibiotics, such as Hydroxyurea, Anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin and other derivatives); Anthracenediones (e.g, Mitoxantrone and relatives); antibiotics from the Streptomyces family (e.g., Bleomycin, Mitomycin C, Actinomycin, Plicamycin); and ultraviolet light.

In certain embodiments, the ATR inhibitor is a compound of Formula A-1, compound of Formula A-2, compound of Formula A-3, compound of Formula A-4, or AZD-6738, and the DNA damaging agent comprises chemotherapy. In certain embodiments, the ATR inhibitor is a compound of Formula A-1, a compound of Formula A-2, a compound of Formula A-3, a compound of Formula A-4, or AZD-6738 and the DNA damaging agent comprises a platinating agent (e.g., cisplatin, carboplatin). In certain embodiments, the ATR inhibitor is a compound of Formula A-1, a compound of Formula A-2, a compound of Formula A-3, a compound of Formula A-4, or AZD-6738 and the DNA damaging agent comprises a antimetabolite (e.g., gemcitabine). In certain embodiments, the ATR inhibitor is a compound of Formula A-2 and the DNA damaging agent is cisplatin or gemcitabine.

In certain embodiments, the ATR inhibitor is a compound of Formula A-1 and the DNA damaging agent is cisplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-2 and the DNA damaging agent is cisplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-3 and the DNA damaging agent is cisplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-4 and the DNA damaging agent is cisplatin or gemcitabine. In certain embodiments, the ATR inhibitor is AZD-6738 and the DNA damaging agent is cisplatin or gemcitabine.

In certain embodiments, the ATR inhibitor is a compound of Formula A-1 and the DNA damaging agent is carboplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-2 and the DNA damaging agent is carboplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-3 and the DNA damaging agent is carboplatin or gemcitabine. In certain embodiments, the ATR inhibitor is a compound of Formula A-4 and the DNA damaging agent is carboplatin or gemcitabine. In certain embodiments, the ATR inhibitor is AZD-6738 and the DNA damaging agent is carboplatin or gemcitabine.

Dosages of DNA Damaging Agent and ATR Inhibitor

In general, any effective dose of an ATR inhibitor and DNA damaging agent may be administered. In some embodiments, an ATR inhibitor (e.g., a compound of Formula A-2) when used in a combination therapy with a DNA-damaging agent, as described herein, may be administered at a dosage of between about 50 mg/m² and about 300 mg/m², between about 50 mg/m² and about 240 mg/m², between about 60 mg/m² and about 240 mg/m², between about 60 mg/m² and about 180 mg/m², between about 60 mg/m² and about 120 mg/m², between about 80 mg/m² and about 120 mg/m², between about 90 mg/m² and about 120 mg/m², or between about 80 mg/m² and about 100 mg/m². In certain embodiments, an ATR inhibitor may be administered at a dosage between about 50 mg/m² and about 300 mg/m² (e.g., about 240 mg/m²). In some instances, an ATR inhibitor may be administered at a dosage between about 60 mg/m² and about 180 mg/m² (e.g., 120 mg/m²). In certain cases, an ATR inhibitor may be administered at a dosage between about 80 mg/m² and about 100 mg/m² (e.g., about 90 mg/m²). In some embodiments, ATR inhibitor (e.g., a compound of Formula A-2) may be administered at a dosage of about 90 mg/m² or about 120 mg/m².

In some embodiments, a platinating agent (e.g., carboplatin) when used in a combination therapy with an ATR inhibitor (e.g., a compound of Formula A-2), as described herein, may be administered at a target AUC of between about 3 mg/mL·min and about 6 mg/mL·min, between about 3.5 mg/mL·min and about 6 mg/mL·min, between about 4 mg/mL·min and about 6 mg/mL·min, between about 4 mg/mL·min and about 5.5 mg/mL·min, or between about 4 mg/mL·min and about 5 mg/mL·min. In some embodiments, a platinating agent (e.g., carboplatin) may be administered at a target AUC of between about 3 mg/mL·min and about 6 mg/mL·min. In certain embodiments, a platinating agent (e.g., carboplatin) may be administered with at a target AUC of between about 4 mg/mL·min and about 5 mg/mL·min. As used herein, the term “target AUC” refers the target area under the plasma concentration versus time curve. The term “AUC” refers the area under the plasma concentration versus time curve. The dosage of certain DNA damaging agents, such as carboplatin, may be determined from the drug label information. For example, the dosage in mg of carboplatin may be determined from the target AUC based on mathematical formula, which is based on a patient's pre-existing renal function or renal function and desired platelet nadir. The Calvert formula, shown below, is used to calculate dosage in milligrams, based upon a patient's glomerular filtration rate (GFR in mL/min) and carboplatin target area under the concentration versus time curve (AUC in mg/mL·min). GFR may be measured using ⁵¹Cr-EDTA clearance or may be estimated using methods known to ordinary skill in the art.

Total Dose (mg)=(target AUC)×(GFR+25)

It should be understood that all combinations of the above-referenced ranges for dosage of ATR inhibitor and dosage of a DNA damaging agent for use in a combination therapy, as described herein, may be possible. For instance, in some embodiments, a platinating agent (e.g., carboplatin) may be administered with at a target AUC of between about 3 mg/mL·min and about 6 mg/mL·min (e.g., between about 4 mg/mL·min and about 6 mg/mL·min, between about 4 mg/mL·min and about 5 mg/mL·min) and an ATR inhibitor (e.g., a compound of Formula A-2) may be administered with at a dosage between about 50 mg/m² and about 300 mg/m² (e.g., between about 60 mg/m² and about 180 mg/m², between about 80 mg/m² and about 100 mg/m²).

In certain embodiments, a platinating agent (e.g., carboplatin) may be administered with at a target AUC of between about 3 mg/mL·min and about 6 mg/mL·min (e.g., between about 4 mg/mL·min and about 6 mg/mL·min) and an ATR inhibitor (e.g., a compound of Formula A-2) may be administered with at a dosage between about 60 mg/m² and about 180 mg/m² (e.g., about 90 mg/m², about 120 mg/m²). In one example, a method of treating a proliferative disorder (e.g., ovarian, lung, colorectal) may comprise administering a platinating agent (e.g., carboplatin) at a target AUC of between about 4 mg/mL·min and about 5 mg/mL·min on day 1, a dose of between about 90 mg/m² and about 120 mg/m² of a first dose of ATR inhibitor (e.g., a compound of Formula A-2) on day 2 between about 20 hours and about 28 hours (e.g., about 24 hours or 24 hours±2 hours) after administration of the platinating agent, and a second dose of the ATR inhibitor between about 6 days and about 8 days (e.g., on day 9) after the first dose. Such a treatment method may lead to at least a RECIST partial response and/or may decrease the sum of the longest diameter of target lesions, decrease the sum of the longest diameter of non-target lesions, and/or decrease tumor burden by between about 20% and about 60%, or between about 40% and about 60%. In some such embodiments, the proliferative disorder (e.g., ovarian cancer, lung cancer, colorectal cancer, breast cancer) may have a defect in ATM signaling (e.g., mutation in p53, partial loss of ATM signaling, complete loss of ATM signaling).

In other embodiments in which the ATR inhibitor (e.g., a compound of Formula A-2) is administered as a monotherapy or a combination therapy with a DNA-damaging agent, as described herein, said ATR inhibitor (e.g., a compound of Formula A-2) may be administered at a dosage of between about 50 mg/m² and about 500 mg/m², between about 100 mg/m² and about 500 mg/m², between about 120 mg/m² and about 500 mg/m², between about 240 mg/m² and about 480 mg/m², between about 50 mg/m² and about 480 mg/m², between about 50 mg/m² and about 300 mg/m², between about 50 mg/m² and about 240 mg/m², or between about 50 mg/m² and about 120 mg/m². In some embodiments, said ATR inhibitor (e.g., a compound of Formula A-2) may be administered at a dosage of about 60 mg/m², about 120 mg/m², about 240 mg/m², or 480 mg/m². In some embodiments, said ATR inhibitor (e.g., a compound of Formula A-2) may be administered at a dosage of about 240 mg/m² or about 480 mg/m² as a monotherapy. In some embodiments, said ATR inhibitor is Compound A-2. In some embodiments, Compound A-2 is administered at a dosage of about 240 mg/m² as a monotherapy. In some embodiments, Compound A-2 is administered at a dosage of about 240 mg/m² as a monotherapy once weekly or twice weekly.

In some embodiments, cisplatin is used in a combination therapy with a compound of Formula A-2, wherein the dosage of cisplatin is at between about 30 and about 90 mg/m², between about 40 and about 75 mg/m², or between about 60 and about 90 mg/m², and wherein the dosage of the compound of Formula A-2 is between about 60 mg/m² and about 240 mg/m², between about 120 mg/m² and 160 mg/m², or between about 90 mg/m² and about 210 mg/m². In some specific embodiments, the dosage of cisplatin is at 40 mg/m², 60 mg/m², or 75 mg/m². In some specific embodiments, the dosage of a compound of Formula A-2 is about 90 mg/m², 140 mg/m², or 210 mg/m². In some specific embodiments, the dosage of cisplatin is at between about 30 and about 90 mg/m², and the dosage of the compound of Formula A-2 is between about 60 mg/m² and about 240 mg/m². In some specific embodiments, the dosage of cisplatin is at between about 40 and about 75 mg/m², and the dosage of a compound of Formula A-2 is between about 90 mg/m² and 210 mg/m². In some specific embodiments, the dosage of cisplatin is at between about 60 and about 90 mg/m², and the dosage of the compound of Formula A-2 is between about 120 mg/m² and 160 mg/m². In some specific embodiments, the dosage of cisplatin is at about 75 mg/m², and the dosage of the compound of Formula A-2 is about 140 mg/m².

In some embodiments, gemcitabine is used in a combination therapy with a compound of Formula A-2, wherein the dosage of gemcitabine is between about 300 and about 1200 mg/m², between about 875 mg/m² and 1125 mg/m², or between about 500 mg/m² and about 1000 mg/m², and wherein the dosage of the compound of Formula A-2 is between about 10 mg/m² and about 240 mg/m², between about 18 mg/m² and 210 mg/m², or between about 180 mg/m² and 240 mg/m². In some specific embodiments, gemcitabine may be administered at 500 mg/m², 750 mg/m², 875 mg/m², or 1000 mg/m². In some specific embodiments, the dosage of the compound of Formula A-2 is about 18 mg/m², 36 mg/m², 60 mg/m², 72 mg/m², 90 mg/m², 140 mg/m², or 210 mg/m². In some specific embodiments, the dosage of gemcitabine is between about 300 and about 1200 mg/m², and the dosage of a compound of Formula A-2 is between about 10 mg/m² and about 240 mg/m². In some specific embodiments, the dosage of gemcitabine is between about 500 and about 1000 mg/m², and the dosage of the compound of Formula A-2 is between about 18 mg/m² and about 210 mg/m². In some specific embodiments, the dosage of gemcitabine is between about 875 mg/m² and about 1125 mg/m², and the dosage of the compound of Formula A-2 is between about 180 mg/m² and about 240 mg/m². In some specific embodiments, the dosage of gemcitabine is about 1000 mg/m², and the dosage of the compound of Formula A-2 is about 210 mg/m².

In some embodiments, gemcitabine and cisplatin are used in a combination therapy with a compound of Formula A-2, wherein the dosage of gemcitabine is between about 300 and about 1200 mg/m², between about 500 mg/m² and 1,000 mg/m², or between about 700 mg/m² and about 1,000 mg/m², and wherein the dosage of the compound of Formula A-2 is between about 10 mg/m² and about 250 mg/m², between about 30 mg/m² and 250 mg/m², or between about 50 mg/m² and 200 mg/m², or between about 80 mg/m² and 200 mg/m², and wherein the dosage of cisplatin is between about 30 mg/m² and 90 mg/m², or between about 50 mg/m² and 90 mg/m². In some specific embodiments, gemcitabine may be administered at 500 mg/m², 750 mg/m², 875 mg/m², or 1000 mg/m². In some specific embodiments, the dosage of the compound of Formula A-2 is about 18 mg/m², 36 mg/m², 60 mg/m², 72 mg/m², 90 mg/m², 140 mg/m², or 210 mg/m². In some specific embodiments, the dosage of cisplatin is about 40 mg/m², 60 mg/m², 75 mg/m², 90 mg/m², 140 mg/m², or 210 mg/m². In some specific embodiments, the dosage of gemcitabine is between about 500 and about 1000 mg/m²; the dosage of the compound of Formula A-2 is between about 60 mg/m² and about 210 mg/m²; and the dosage of cisplatin is between about 50 mg/m² and about 90 mg/m². In some specific embodiments, the dosage of gemcitabine is about 875 mg/m²; the dosage of the compound of Formula A-2 is about 90 mg/m²; and the dosage of cisplatin is about 60 mg/m².

Biomarkers

In some embodiments, one or more biomarkers may be used to monitor or determine the efficacy of the treatment. In certain embodiments, the percentage of phosphorylated Chk1 (i.e., pChk1) in paired samples may be used as a biomarker; pChk1 is believed to correlate with the level of ATR activity. For instance, in some embodiments, the amount of pChk1 in the nuclei of cancer cells/mm² of a tumor biopsy may be used to determine the efficacy of monotherapy with an ATR inhibitor or combination therapy including an ATR inhibitor in a subject. In some such embodiments, a first tumor biopsy may be taken between about one to about three hours (e.g., two hours) before administration of the ATR inhibitor and a second tumor biopsy may be taken between about one to about three hours (e.g., two hours) after administration of the ATR inhibitor. The amount of pChk1 in the nuclei of cancer cells/mm² tumor in the first biopsy is set to be 100%, such that the amount of pChk1 in the nuclei of cancer cells/mm² tumor in the second biopsy is normalized by the amount in the first biopsy.

In some embodiments, a method of treating a proliferative disorder comprising administering a platinating agent (e.g., carboplatin) at a target AUC of between about 4 mg/mL·min and about 5 mg/mL·min on day 1, a dose of between about 90 mg/m² and about 120 mg/m² of a first dose of a ATR inhibitor (e.g., a compound of Formula A-2) between about 20 hours and about 28 hours (e.g., about 24 hours or 24 hours±2 hours) after administration of the platinating agent on day 1, and a second dose of the ATR inhibitor between about 6 days and about 8 days (e.g., on day 9) after the first dose, may substantially reduce the percentage of pChk1 in the nuclei of cancer cells/mm². In some such embodiments, the percentage of pChk1 in the nuclei of cancer cells/mm² may be less than about 40%, less than about 30%, less than about 20%, or less than about 10% between about one to about three hours (e.g., two hours) after administration of the ATR inhibitor.

In some embodiments, certain cancer specific biomarkers may be used to monitor or determine the efficacy of the treatment. For instance, CA125 ovarian cancer tumor burden marker may be used to assess treatment of ovarian cancer with monotherapy or with combination therapy including an ATR inhibitor and a DNA damaging agent.

In some embodiments, certain treatment related adverse events in a subject may be used as biomarkers to monitor or determine the efficacy of the treatment. In certain embodiments, an adverse event may be indicative of the mechanism of action of the ATR inhibitor in a combination therapy. For example, the presence of neutropenia and thrombocytopenia may be used as biomarkers.

Pharmaceutically Acceptable Salts, Solvates, Chlatrates, Prodrugs and Other Derivatives

The compounds described herein can exist in free form, or, where appropriate, as salts. Those salts that are pharmaceutically acceptable are of particular interest since they are useful in administering the compounds described below for medical purposes. Salts that are not pharmaceutically acceptable are useful in manufacturing processes, for isolation and purification purposes, and in some instances, for use in separating stereoisomeric forms of the compounds of the invention or intermediates thereof.

As used herein, the term “pharmaceutically acceptable salt” refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds.

Where the compound described herein contains a basic group, or a sufficiently basic bioisostere, acid addition salts can be prepared by 1) reacting the purified compound in its free-base form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. In practice, acid addition salts might be a more convenient form for use and use of the salt amounts to use of the free basic form.

Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

Where the compound described herein contains a carboxy group or a sufficiently acidic bioisostere, base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. In practice, use of the base addition salt might be more convenient and use of the salt form inherently amounts to use of the free acid form. Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), alkaline earth metal (e.g., magnesium and calcium), ammonium and N⁺(C₁₋₄alkyl)₄ salts. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization.

Basic addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum. The sodium and potassium salts are usually preferred. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate. Suitable inorganic base addition salts are prepared from metal bases, which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N′-dibenzylethylenediamine, chloroprocaine, dietanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, dicyclohexylamine and the like are examples of suitable base addition salts.

Other acids and bases, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds described herein and their pharmaceutically acceptable acid or base addition salts.

It should be understood that this invention includes mixtures/combinations of different pharmaceutically acceptable salts and also mixtures/combinations of compounds in free form and pharmaceutically acceptable salts.

The compounds described herein can also exist as pharmaceutically acceptable solvates (e.g., hydrates) and clathrates. As used herein, the term “pharmaceutically acceptable solvate,” is a solvate formed from the association of one or more pharmaceutically acceptable solvent molecules to one of the compounds described herein. The term solvate includes hydrates (e.g., hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, and the like).

As used herein, the term “hydrate” means a compound described herein or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

As used herein, the term “clathrate” means a compound described herein or a salt thereof in the form of a crystal lattice that contains spaces (e.g., channels) that have a guest molecule (e.g., a solvent or water) trapped within.

In addition to the compounds described herein, pharmaceutically acceptable derivatives or prodrugs of these compounds may also be employed in compositions to treat or prevent the herein identified disorders.

A “pharmaceutically acceptable derivative or prodrug” includes any pharmaceutically acceptable ester, salt of an ester, or other derivative or salt thereof of a compound described herein which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound described herein or an inhibitorily active metabolite or residue thereof. Particularly favoured derivatives or prodrugs are those that increase the bioavailability of the compounds when such compounds are administered to a patient (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species.

As used herein and unless otherwise indicated, the term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound described herein. Prodrugs may become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds of the invention that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds described herein that comprise —NO, —NO₂, —ONO, or —ONO₂ moieties. Prodrugs can typically be prepared using well-known methods, such as those described by Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed).

Therapeutic Uses

The present disclosure provides a method of treating diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation, including proliferative or hyperproliferative diseases, in a subject. A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.

The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.

The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, hematological malignancies. The term “hematological malignancy” refers to tumors that affect blood, bone marrow, and/or lymph nodes. Exemplary hematological malignancies include, but are not limited to, leukemia, such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL, e.g., activated B-cell (ABC) DLBCL (ABC-DLBCL))), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, Waldenström's macroglobulinemia (WM, lymphoplasmacytic lymphoma), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, central nervous system (CNS) lymphoma (e.g., primary CNS lymphoma and secondary CNS lymphoma); and T-cell NHL, such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); lymphoma of an immune privileged site (e.g., cerebral lymphoma, ocular lymphoma, lymphoma of the placenta, lymphoma of the fetus, testicular lymphoma); a mixture of one or more leukemia/lymphoma as described above; myelodysplasia; and multiple myeloma (MM). Additional exemplary cancers include, but are not limited to, lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, a.k.a. Wilms' tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor, neuroendocrine prostate); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; adenoid cystic carcinoma; and vulvar cancer (e.g., Paget's disease of the vulva).

The term “inflammatory disease” refers to a disease caused by, resulting from, or resulting in inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis. An ocular inflammatory disease includes, but is not limited to, post-surgical inflammation.

An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

In some embodiments, the term “cancer” includes, but is not limited to the following types of cancers: oral, lung, gastrointestinal, genitourinary tract, liver, bone, nervous system, gynecological, skin, thyroid gland, or adrenal gland. More specifically, “cancer” includes, but is not limited to the following cancers: oral cancers, such as buccal cavity cancer, lip cancer tongue cancer, mouth cancer, and pharynx cancer; cardiac cancers: sarcoma (e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma; lung cancers, such as bronchogenic carcinoma (e.g., squamous cell or epidermoid, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (e.g., bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous, hamartoma, and mesothelioma; gastrointestinal cancers, such as esophageal cancer (e.g., squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach cancer (e.g., carcinoma, lymphoma, leiomyosarcoma), pancreatic cancer (e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or small intestinal cancer (e.g., adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel or large intestinal cancer (e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), colon cancer, colon-rectum cancer, colorectal cancer, and rectum cancer, genitourinary tract cancers, such as kidney cancer (e.g., adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder cancer, urethral cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (e.g., adenocarcinoma, sarcoma), and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver cancers, such as hepatoma (e.g., hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, and biliary passages; bone cancers, such as osteogenic sarcoma (e.g., osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (e.g., osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, and osteoid osteoma and giant cell tumors; Nervous system cancers, such as: skull cancer (e.g., osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meningeal cancer (e.g., meningioma, meningiosarcoma, gliomatosis), brain cancer (e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cord cancer (e.g., neurofibroma, meningioma, glioma, sarcoma); gynecological cancers: uterine cancer (e.g., endometrial carcinoma), cervical cancer (e.g., cervical carcinoma, pre-tumor cervical dysplasia), ovarian cancer (e.g., ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulvar cancer (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (e.g., clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube cancer (e.g., carcinoma), an breast cancer; hematologic cancers, such as blood cancer (e.g., myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell, and lymphoid disorders; skin cancers, such as malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; thyroid gland cancers, such as papillary thyroid carcinoma, follicular thyroid carcinoma, undifferentiated thyroid cancer, medullary thyroid carcinoma, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma, and Adenoid cystic carcinoma; and adrenal gland cancers, such as neuroblastoma.

In other embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma), head and neck cancer (e.g., nasopharyngeal cancer), pancreatic cancer, breast cancer (e.g., triple negative breast cancer), gastric cancer, brain cancer, endometrial carcinoma, pancreatic cancer, biliary tract cancer, bladder cancer, colorectal cancer, glioblastoma, esophageal cancer, hepatocellular carcinoma, neuroendocrine cancer, or ovarian cancer. In certain embodiments, the cancer is lung cancer (e.g., mesothelioma), breast cancer (e.g., triple negative breast cancer), neuroendocrine cancer (e.g., neuroendocrine prostate cancer), or ovarian cancer (e.g., CA 125 positive ovarian cancer). In certain embodiments, the cancer is nasopharyngeal cancer. In certain embodiments, the cancer is fallopian tube cancer, peritoneal cancer, urothelial carcinoma, esophageal cancer, or head and neck squamous cell carcinoma.

The method comprises administering to a subject in need thereof a DNA damaging agent, and between about 12-48 hours later administering to the subject a compound that inhibits ATR protein kinase. In some embodiments, the compound that inhibits ATR is administered about 18-42 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-40 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 12-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 18-36 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 20-28 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered about 24 hours after administration of the DNA damaging agent. In some embodiments, the compound that inhibits ATR is administered 24 hours±2 hours after administration of the DNA damaging agent. In some embodiments, said DNA-damaging agent is chemotherapy or radiation treatment.

A “subject” to which administration is contemplated includes, but is not limited to, humans; commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys). A subject in need of treatment is a subject identified as having a proliferative disorder i.e., the subject has been diagnosed by a physician (e.g., using methods well known in the art) as having a proliferative disorder (e.g., a cancer). In some embodiments, the subject in need of treatment is a subject suspected of having or developing a proliferative disorder, such as a subject presenting one or more symptoms indicative of a proliferative disorder. The term “subject in need of treatment” further includes people who once had a proliferative disorder but whose symptoms have ameliorated. For cancer, the one or more symptoms or clinical features depend on the type and location of the tumor. For example, lung tumors may cause coughing, shortness of breath, or chest pain. Tumors of the colon can cause weight loss, diarrhea, constipation, iron deficiency anemia, and blood in the stool. The following symptoms occur with most tumors: chills, fatigue, fever, loss of appetite, malaise, night sweats, and weight loss.

The terms “administer,” “administering,” or “administration,” as used herein refers to implanting, absorbing, ingesting, injecting, or inhaling the one or more therapeutic agents.

As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of proliferative disorder. In some embodiments, treatment may be administered after one or more signs or symptoms have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the proliferative disorder. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

As used herein, the terms “tumor burden” has its ordinary meaning in the art and may refer to the number of cancer cells, the size of a tumor, or the amount of cancer in the body.

As used herein, the terms “about” has its ordinary meaning in the art. In some embodiments with respect to time, about may be within 50 minutes, within 40 minutes, within 30 minutes, within 20 minutes, within 10 minutes, within 5 minutes, or within 1 minute of the specified time. In some embodiments with respect to dosage, about may be within 20%, within 15%, within 10%, within 5%, or within 1% of the specified dosage.

An “effective amount” refers to an amount sufficient to elicit the desired biological response, i.e., treating the proliferative disorder. As will be appreciated by those of ordinary skill in this art, the effective amount of the compounds described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount includes, but is not limited to, that amount necessary to slow, reduce, inhibit, ameliorate or reverse one or more symptoms associated with neoplasia. For example, in the treatment of cancer, such terms may refer to a reduction in the size of the tumor.

An effective amount of a compound may vary from about 0.001 mg/kg to about 1000 mg/kg in one or more dose administrations, for one or several days (depending on the mode of administration). In certain embodiments, the effective amount varies from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, and from about 10.0 mg/kg to about 150 mg/kg.

The compounds provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. The desired dosage can be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.

In certain embodiments, the compounds provided herein may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

Biological Samples

As inhibitors of the ATR pathway, the compounds and compositions of this invention are also useful in biological samples. One aspect of the invention relates to inducing DNA damage and inhibiting ATR in a biological sample, which method comprises contacting said biological sample with a DNA damaging agent followed by contacting the sample about 12-48 hours later with a compound that inhibits ATR kinase activity. The term “biological sample”, as used herein, means an in vitro or an ex vivo sample, including, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.

Inducing DNA damage followed by inhibition of ATR activity in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, blood transfusion, organ-transplantation, and biological specimen storage.

EXAMPLES Example 1. Optimization of Dose Schedule In Vitro

The effect of altering a compound of Formula A-2 (Compound A-2) dosing schedule in combination with DNA damaging agents was assessed with gemcitabine in a pancreatic cancer cell line (PSN1) as shown in FIG. 1 . Cells were treated with gemcitabine for 24 hours in combination with Compound A-2; Compound A-2 was added for 2 hour periods at various times both during and after gemcitabine treatment. Cell viability was measured by MTS assay (96 hours) and the data subjected to a statistical Bliss analysis. Compound A-2 synergized with gemcitabine when administered at the start of gemcitabine treatment. This synergistic effect markedly increased as Compound A-2 was administered progressively later through the 24 hour gemcitabine dosing period. Synergy was maximal when Compound A-2 was administered 24 hours after starting gemcitabine treatment as shown in FIG. 1 ; later administration of Compound A-2 was less effective. No synergy was seen when Compound A-2 was administered 48 hours or later, after gemcitabine treatment was started. The strong schedule dependence is attributed to an accumulation of cells in S phase, and concomitant increase in ATR activity (measured by P-Chk1) that occurs in response to gemcitabine treatment alone. Thus, maximal impact of Compound A-2 is expected at a time when most cells are in S phase as a result of gemcitabine treatment. Extended intervals (>48 hours) between gemcitabine therapy and Compound A-2 exposure allows DNA damage to be repaired, permitting cells to exit S phase and dramatically reducing the impact of ATR inhibition.

These data indicate that short exposures to Compound A-2 are sufficient to sensitize certain cancer cells to DNA damaging agents. Furthermore, the data suggest that administering Compound A-2 after treatment with a DNA damaging agent, to coincide with maximal S phase accumulation is optimal.

Example 2: Dose Schedule Optimization of a Compound of Formula A-2 in Combination with Gemcitabine or Cisplatin

In vitro pharmacology studies have demonstrated that combinations of a compound of Formula A-2 (Compound A-2) and DNA damaging drugs are most effective when cells are treated with Compound A-2 after the DNA damaging drug, and when Compound A-2 addition is timed to coincide with peak accumulation of cells in S phase. The optimal in vivo dose schedule for Compound A-2 was assessed in combination with gemcitabine and cisplatin in 2 separate xenograft models.

In a human pancreatic cancer xenograft model (PSN1), Compound A-2 (20 mg/kg, dosed every 6 days) was most effective when dosed 12 to 24 hours after administration of gemcitabine (15 mg/kg, dosed every 3 days) Dosing with Compound A-2the within 12 hours of gemcitabine administration, or beyond 24 hours of gemcitabine administration, reduced efficacy. FIG. 2 shows the Compound A-2 dose schedule in combination with gemcitabine in vivo.

Dosing with Compound A-2 before gemcitabine, or 48 hours after gemcitabine, provided limited benefit over gemcitabine treatment. The addition of Compound A-2 to gemcitabine did not lead to increased body weight loss when compared with gemcitabine only treatment.

In a NSCLC xenograft model derived from primary human tumor tissue, in SCID mice, Compound A-2the (10 mg/kg, dosed twice weekly) was most effective when dosed 14 hours after administration of cisplatin (3 mg/kg, dosed weekly) as shown in FIG. 3 . Under these conditions, marked tumor regression (43%) and substantial growth delay was observed. This contrasts with the effects of either agent given alone, where neither Compound A-2the nor cisplatin had any meaningful impact on tumor growth (≤10% tumor growth inhibition). This combination was well tolerated with <2% body weight loss at nadir, though animals did not gain weight as rapidly as cisplatin only treated animals.

These studies demonstrate strong schedule dependence for intravenously administered Compound A-2. For combinations with either gemcitabine or cisplatin, efficacy was maximal when Compound A-2 was dosed 12 to 24 hours after the DNA-damaging agent.

Example 3—Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 as Monotherapy (Mono) or in Combination with Carboplatin (CP) in Advanced Cancer Patients (Pts) with Preliminary Evidence of Target Modulation and Antitumor Activity

ATR mediates the homologous recombination DNA repair pathway and cellular response to replication stress. The compound of Formula A-2 (Compound A-2) is a potent and selective inhibitor of ATR (Ki<0.2 nM) that showed enhanced synergy of ATR inhibition with cytotoxic chemotherapy, and potential monotherapy ATR inhibitor activity in tumor cell lines with high levels of replication stress, such as defects in the DNA damage repair (DDR) pathway (e.g. ATM loss). A Phase I dose-escalation trial of Compound A-2 was undertaken to assess the safety and tolerability of an ATR inhibitor as a monotherapy and in combination with DNA-damaging chemotherapy, to show evidence of ATR inhibition in tumor tissue, and to explore antitumor activity.

Pts with advanced solid tumors enrolled in 2 sequential parts. In Part A, pts received IV Compound A-2 mono weekly in single-pt cohorts, with 3+3 cohorts initiated if grade (G) ≥2 Compound A-2 related adverse events (AEs) were observed. In Part B, pts received CP on day 1 and Compound A-2 on days 2 and 9 of a 21-day cycle in a 3+3 dose-escalation design (Compound A-2 on days 2 and 9 every 3 weeks and CP on day 1 every 3 weeks). Paired Compound A-2 tumor biopsies were obtained in selected CP treated pts pre- and post-Compound A-2, and pS345 Chk1 levels were assessed by immunohistochemistry (IHC).

Results: 25 pts were treated; M/F 10/15; median age 67 yr (range 49-76 yr); ECOG PS 0/1:11/14. In Part A, 11 pts (colorectal [CRC; n=2]; mesothelioma [n=2]; other [n=7]; median prior lines of therapy=3) received Compound A-2 at 60 mg/m² (n=1), 120 mg/m² (n=2), 240 mg/m² (n=1) and 480 mg/m² (n=7). In Part B, 14 pts (CRC [n=6]; ovarian [n=2]; other [n=6]; median prior lines of therapy=3) received Compound A-2 240 mg/m²+CP AUC 5 mg/mL·min (n=3; dose level 1 [DL1]), Compound A-2 120 mg/m²+CP AUC 5 mg/mL·min (n=3; DL2), Compound A-2 120 mg/m²+CP AUC 4 mg/mL·min (n=3; DL3) and Compound A-2 90 mg/m²+CP AUC 5 mg/mL·min (n=5; DL4). In Part A, no dose-limiting toxicities (DLT) or drug-related G3-4 AEs were seen. In Part B, 2 pts had DLT: G4 neutropenia and fever (n=1; DL1) and G3 hypersensitivity (n=1; DL2). Non-DLT G3-4 AEs were neutropenia (n=4; DL1-2) and thrombocytopenia (n=1; DL2) requiring dose delays. No G3-4 AEs were seen at DL3-4. RP2D cohort expansion is ongoing at DL4. Compound A-2 displayed linear AUC and C_(max) at all DLs; median half-life was 16h with no accumulation. Based on preclinical models, efficacious exposures were achieved. When combined with CP, DL1 and DL2 showed similar Compound A-2 exposure, suggesting no apparent drug interactions. Decreased Chk1 phosphorylation was seen in 2/2 paired tumor biopsies (74% at DL4; 94% at DL2). An advanced CRC pt (serosal disease and abdominal lymphadenopathy; 3 prior lines of chemotherapy) with complete ATM loss by IHC achieved RECIST complete response to Compound A-2 mono at 60 mg/m² and remains on trial at 59+ wks. RECIST stable disease (SD) was seen with Compound A-2 mono in 4 pts (median duration of SD=11 wks [11-17.4 wks]) and Compound A-2+CP in 7 pts, who were still ongoing (duration of SD=5+ to 20+ wks), including several pts who had progressed on prior platinum therapy.

Example 4. Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 as Monotherapy (Mono)

This example further describes the Phase I dose-escalation trial of a compound of Formula A-2 (Compound A-2) to assess the safety and tolerability of an ATR inhibitor as a monotherapy in Example 3.

The study included 17 patients. The subject demographic and baseline characteristics are shown in Table 1.

TABLE 1 Subject demographics and baseline characteristics. Baseline Characteristics Age, mean (SD), years 63.4 (10.3) Sex, n (%) Male 7 (41.2) Female 10 (58.8) Race, n (%) White 16 (94.1) Black or African American 0 Asian 0 Other 1 (5.9) ECOG PS at baseline, n (%) 0 4 (23.5) 1 13 (76.5) Primary malignancy, n (%) NSCLC 0 Breast cancer 1 (5.9) Ovarian cancer 1 (5.9) Colorectal cancer 3 (17.6) Other 12 (70.6)

The 17 subjects received once weekly intravenous doses of Compound A-2 ranging from 60 to 480 mg/m² or twice weekly intravenous doses of 240 mg/m² of Compound A-2. Eleven of the subjects were given a once weekly dosage of Compound A-2 as a monotherapy. Six of the subjects were given a twice weekly dosage of Compound A-2 as a monotherapy. For the once weekly dosage, there were no dose limiting toxicities (DLTs) and adverse events began at a dose of 480 mg/m² as shown in Table 2. DLTs were defined according to the National Cancer Institute (NCI) CTCAE (Version 4). FIG. 12 shows tumor response showing changes from baseline in cancer subjects who received treatment of Compound A-2 as a monotherapy, and FIG. 13 shows duration of progression-free survival (PFS).

TABLE 2 Compound A-2 monotherapy weekly treatment related adverse events mg/m² 60 Once 120 Once 240 Once 480 Once 240 Twice Weekly Weekly Weekly Weekly Weekly Total (n = 1) (n = 2) (n = 1) (n = 7) (n = 6) (N = 17) n (%) n (%) n (%) n (%) n (%) n (%) Grade ≥ All Grade ≥ All Grade ≥ All Grade ≥ All Grade ≥ All Grade ≥ All Event 3 Grades 3 Grades 3 Grades 3 Grades 3 Grades 3 Grades Any 0 1 (100) 0 4 (50.0) 0 0 0 7 (100) 1 (16.7) 6 (100) 1 (5.9) 15 (88.2) Event Flushing 0 0 0 0 0 0 0 3 (42.9) 0 1 (16.7) 0  4 (23.5) Catheter 0 0 0 0 0 0 0 0 0 2 (33.3) 0  2 (11.8) site related reaction Nausea 0 0 0 0 0 0 0 2 (28.6) 0 0 0  2 (11.8) Pruritus 0 0 0 0 0 0 0 2 (28.6) 0 0 0  2 (11.8) Infusion 0 0 0 0 0 0 0 1 (14.3) 0 4 (16.7) 0  2 (11.8) related reaction Headache 0 0 0 0 0 0 0 2 (28.6) 0 0 0  2 (11.8)

As noted in Example 3, a subject with KRAS and BRAF wildtype metastatic colorectal cancer with serosal disease and abdominal lymphadenopathy had a RECIST complete response after treatment with 60 mg/m² monotherapy (weekly) of Compound A-2. The colorectal cancer had a complete loss of ATM signaling based on IHC analysis. There was loss of MLH1 and PMS2, and weak heterogeneous staining of MSH2 and MSH6 on immunohistochemistry. Targeted and whole exome next-generation sequencing (NGS) revealed a somatic truncating mutation in MLH1 at position p.Lys33*/c.97A>T, which was likely to contribute to tumor microsatellite instability (MSI). PTEN and CTNNB1 somatic mutations were also detected on NGS. The subject remains on trial with ongoing RECIST complete response lasting more than 28 months. Radiographs of the left common iliac lymph node before treatment (i.e., baseline) and after 15 months of treatment is shown in FIG. 4 .

Prior to treatment with Compound A-2, the subject received 4 lines of treatment. The first line of treatment was folinic acid, Fluorouracil, irinotecan, and cetuximab, which resulted in a RECIST partial response. The second line of treatment was folinic acid, fluorouracil, oxaliplatin, and Avastin, which resulted in a RECIST partial response. The third line of treatment was fluorouracil and Avastin, which resulted in a RECIST progression. The fourth line of treatment was Capecitabine and mitomycin which resulted in a RECIST progression.

The tumor response and duration of progression free survival for several cancers are shown in FIGS. 12 and 13 , respectively.

Example 5. Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 in Combination with Carboplatin (CP) in Advanced Cancer Patients (Pts) with Preliminary Evidence of Target Modulation and Antitumor Activity

This example further describes the Phase I dose-escalation trial of a compound of Formula A-2 (Compound A-2) in combination with carboplatin in Example 3.

The study included 19 patients. The subject demographic and baseline characteristics are shown in Table 3.

TABLE 3 Subject demographics and baseline characteristics. Part B Baseline Characteristics (N = 19) Age, mean (SD), years 63.3 (8.7) Sex, n (%) Male 7 (36.8) Female 12 (63.2) Race, n (%) White 18 (94.7) Black or African American 0 Asian 0 Other 1 (5.3) ECOG PS at baseline, n (%) 0 7 (36.8) 1 12 (63.2) Primary malignancy, n (%) NSCLC 1 (5.3) Breast cancer 1 (5.3) Ovarian cancer 2 (10.5) Colorectal cancer 8 (42.1) Other 7 (36.8)

The 15 subjects received a three week cycle of carboplatin on day 1, Compound A-2 on day 2, 24 hours after treatment with carboplatin, and Compound A-2 on day 9. The dose escalation and dose limiting toxicities are shown in Table 4.

TABLE 4 Dose escalation of Compound A-2 combination therapy. Carboplatin No. Subjects A-2 Dose Dose (Enrolled/ Cohort (mg/m²) (AUC) DLT evaluable) DLTs 1 240 5 mg/mL · min 3/3 1 (febrile neutropenia) 2 120 5 mg/mL · min 3/3 1 (acute hypersensitivity) 3 120 4 mg/mL · min 3/3 0 4 90 5 mg/mL · min 10/10 1 (febrile neutropenia)

The adverse events are shown in Tables 5A-5B and include neutropenia and thrombocytopenia, which were believed to be a result of the mechanism of action of ATR when administered in combination with a DNA damaging agent.

TABLE 5A Treatment related adverse events for combination therapy. A-2 (mg/m²) + 240 + 120 + 120 + 90 + Carboplatin AUC5 AUC5 AUC4 AUC5 Total (AUC, mg/mL · min) (N = 3) (N = 3) (N = 3) (N = 6) (N = 15) Adverse Event ≥3 All ≥3 All ≥3 All ≥3 All ≥3 All Anemia 0 3 0 2 0 1 0 3 0 9 Neutropenia 2 3 1 2 0 0 1 2 4 7 Thrombocytopenia 1 3 0 1 0 1 0 2 1 7 Nausea 0 3 0 2 0 1 0 1 0 7 Fatigue 0 2 0 1 0 0 0 3 0 6 Flushing 0 1 1 1 0 1 0 0 1 3 Vomiting 0 2 0 0 0 0 0 0 0 2 Alopecia 0 0 0 0 0 0 0 2 0 2 Headache 0 2 0 0 0 0 0 0 0 2 Neuropathy peripheral 0 2 0 0 0 0 0 0 0 2 Hypertension 1 1 0 0 0 0 0 0 1 1 Respiratory tract 1 1 0 0 0 0 0 c 1 1 infection

TABLE 5B Treatment related adverse events for combination therapy. A-2 (mg/m2) + Carboplatin (AUC) 240 + 5 120 + 5 120 + 4 90 + 5 Total (n = 3) (n = 3) (n = 3) (n = 3) (N = 19) n (%) n (%) n (%) n (%) n (%) Grade ≥ All Grade ≥ All Grade ≥ All Grade ≥ All Grade ≥ All Event 3 Grades 3 Grades 3 Grades 3 Grades 3 Grades Any Event 2 (6 

 .7) 3 (100) 2 (6 

 .7) 3 (100) 0 2 (6 

 .7) 1 (10.0) 9 (90.0) 5 (26.3) 17 (29. 

 ) Anemia 0 3 (100) 0 2 (6 

 .7) 0 1 (33.3) 0 4 (40.0) 0 10 (52. 

 ) Neutropenia 2 (6 

 .7) 3 (100) 1 (33.3) 2 (6 

 .7) 0 0 1 (10.0) 3 (30.0) 4 (21.1) 8 (42.1) Nausea 0 3 (100) 0 2 (6 

 .7) 0 1 (33.3) 0 3 (30.0) 0

  ( 

 7. 

 ) Thrombocytopenia 1 (33.3) 3 (100) 0 1 (33.3) 0 1 (33.3) 0 2 (20.0) 1 (5.3) 7 (3 

 . 

 ) Fatigue 0 2 (6 

 .7) 0 1 (33.3) 0 0 0 4 (40.0) 0 7 (3 

 . 

 ) Pruritus 0 1 (33.3) 0 0 0 1 (33.3) 0 1 (10.0) 0 3 (15. 

 ) Flushing 0 1 (33.3) 0 0 0 1 (33.3) 0 1 (10.0) 0 3 (15. 

 ) Vomiting 0 2 (6 

 .7) 0 0 0 0 0 0 0 2 (1 

 .5) Infusion site  

0 1 (33.3) 0 1 (33.3) 0 0 0 0 0 2 (1 

 .5) Alopecia 0 0 0 0 0 0 0 2 (20.0) 0 2 (1 

 .5) Headache 0 2 (6 

 .7) 0 0 0 0 0 0 0 2 (1 

 .5) Neuropathy peripheral 0 2 (6 

 .7) 0 0 0 0 0 0 0 2 (1 

 .5) Hypersensitivity 0 1 (33.3) 1 (33.3) 1 (33.3) 0 0 0 0 1 (5.3) 2 (1 

 .5) Infusion related reaction 0 0 0 1 (33.3) 0 0 0 1 (10.0) 0 2 (1 

 .5)

indicates data missing or illegible when filed

The Compound A-2 plasma concentration-time profiles were similar with monotherapy and in combination with carboplatin as shown in FIG. 5A and FIG. 5B, respectively. This suggested that there was no interaction of Compound A-2 and carboplatin. In addition, exposure (AUC_(inf) and C_(max)) increased proportionally with increasing dose. Terminal elimination half-life (t_(1/2)) was about 16 hours.

As noted in Example 3, phosphorylated Chk1 was used as a biomarker in paired tumor biopsies of three subjects to determine the efficacy of the combination therapy. A first tumor biopsy was taken on day 2, two hours before Compound A-2 administration, and a second tumor biopsy was taken two hours after Compound A-2 administration. The amount of pChk1 in the nuclei of cancer cells/mm² tumor in the first biopsy was set to 100% and used to normalize the amount in the second biopsy. As shown in FIG. 6 , subject 1 was given target AUC of 5 mg/mL·min of carboplatin and 120 mg/m² of Compound A-2 and had a 94% decrease in pChk1. Subjects 2 and 3 were given target AUC of 5 mg/mL·min of carboplatin and 90 mg/m² of Compound A-2 and had a 73% decrease in pChk1.

As noted in Example 3, a subject with platinum and antimetabolite refractory metastatic high grade serous ovarian cancer having gBRCA1 Q1111Nfs*5 mutation and TP53 Y220C missense deleterious somatic mutation with peritoneal, liver and nodal disease had a RECIST partial response after combination treatment target AUC of 5 mg/mL·min of carboplatin on day 1 and 90 mg/m² of Compound A-2 on day 2, 24 hours after treatment with carboplatin, and 90 mg/m² of Compound A-2 on day 9. The subject remains on trial with ongoing RECIST partial response lasting more than 6 months. Radiographs of left peritoneal disease before treatment (i.e., baseline) and after 5 months of treatment is shown in FIG. 7 .

Prior to treatment with Compound A-2, the subject had debulking surgery before receiving 7 lines of treatment. The seventh line of treatment was carboplatin and gemcitabine, which resulted in progressive disease after 3 cycles. Other lines of treatment included Talazoparib (BMN 673; Biomarin Pharmaceuticals) monotherapy, which resulted in progressive disease after 10 months; Olaparib (AstraZeneca) and AKT inhibitor AZD5363 (AstraZeneca), which resulted in progressive disease after 5 months; and αFR inhibitor, which resulted in progressive disease after 5 months.

The tumor response and duration of progression free survival for several cancers are shown in FIGS. 14 and 15 , respectively.

Tumor response of subjects 1, 2, and 3 shown in FIG. 6 is summarized below:

Treatment Compound A-2 Compound A-2 120 mg/m² + 90 mg/m² + carboplatin AUC carboplatin AUC 5 mg/mL · min 5 mg/mL · min Tumor Ovarian Ovarian Breast (Subject 1) (Subject 2) (Subject 3) PFS (progression- free 175 176 67 survival) (days) Response SD (Stable PR (Partial SD (Stable Disease) Response) Disease)

Example 6. Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 as a Monotherapy (Mono) or in Combination with Carboplatin (CP) in Advanced Cancer Patients (Pts)

ATR mediates the homologous recombination DNA repair pathway and cellular response to replication stress. A compound of Formula A-2 (Compound A-2) is a potent, selective inhibitor of ATR that enhanced ATR inhibition with cytotoxic chemotherapy and inhibited ATR in tumor cell lines with DNA repair pathway defects. Pharmacokinetics (PK), pharmacodynamics (PD), tolerability, and efficacy of a compound of Formula A-2±CP were assessed in a Phase I dose escalation trial.

Patients (pts) with advanced solid tumors measurable by RECIST 1.1 were enrolled in 2 parts. In part A, single pt cohorts received a compound of Formula A-2 once weekly in 21-day cycles with 3+3 dose escalation cohorts being implemented if grade 2 drug-related adverse events (AEs) occurred. In part B, 3+3 pt cohorts received CP on day 1+a compound of Formula A-2 on days 2 and 9 of each 21-day cycle. If no dose limiting toxicities (DLTs) occurred after 1 cycle, dose escalation was permitted in a new cohort. If a DLT was reported, the cohort was expanded to include 3 additional pts with subsequent dose escalation being permitted if no further DLTs occurred. Paired pre- and post-treatment biopsies from selected pts in part B were assessed for pCHK1 levels by immunohistochemistry. Models based on preclinical and clinical data were used to simulate hematologic toxicities and pCHK1 inhibition for pts treated with a compound of Formula A-2 and CP.

Twenty six (26) pts (10 males and 16 females) of median age, 68 years (range 49-76 years) in A and 65 yrs (range 49-74 yrs) in B, with Eastern Cooperative Oncology Group (ECOG) performance status 0/1:9/17 were treated according to the above protocols using the dosage levels (DLs) shown in the table below.

A compound of CP dose Formula A-2 dose (AUC, mg/ # pts DL (mg/m²) mL · min) treated A1 60 — 1 A2 120 — 2 A3 240 — 1 A4 480 — 7 B1 240 5 3 B2 120 5 3 B3 120 4 3 B4 90 5 6

No DLTs occurred in part A. In part B, DLTs were: grade 3/4 neutropenia (2 pts at dosage levels B1 and B2) and grade 3 hypersensitivity (1 pt at dosage level B2). Grade 3/4-related AEs occurred in 5 pts in part B across the various DLs, while none occurred in part A. The best responses were complete response (CR (n=1)) in part A and partial response (PR (n=1)) in part B. The pharmacokinetics (PK) for a compound of Formula A-2 was dose proportional both as a monotherapy and in combination with CP. Based on the above data, the recommended Phase II doses were established at DLs A4 and B4, with modeling predicting 73% pCHK1 inhibition and 5% and <1% probabilities of grade 4 neutropenia and thrombocytopenia, respectively, at DL B4. A compound of Formula A-2 exposures at B4 also led to tumor regression in preclinical models.

Example 7. Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 in Combination with Cisplatin (CIS) in Patients (Pts) with Advanced Solid Tumors

In this Example, patients (pts) received intravenous a compound of Formula A-2 (Compound A-2) in combination with CIS using a 3+3 dose escalation design. CIS was administered on day 1 and a compound of Formula A-2 was administered on days 2 and 9 in 21-day cycles. Twenty eight (28) pts (12 males and 16 females) of median age, 62.5 yrs (range 28-79 yrs), with Eastern Cooperative Oncology Group (ECOG) performance status 0-1 were treated. Primary tumors were breast (n=4), colorectal (n=3), ovarian (n=3), pancreatic (n=2), non-small cell lung cancer (NSCLC) (n=1), and other cancers (n=15). The following table describes the dosage levels (DLs) and various parameters for the various treatment cohorts.

Dose levels were:

No. pts treated/ No. pts evaluable Compound Cisplatin for dose limiting A-2 dose dose toxicities Cohort (mg/m²) (mg/m²) (DLTs) No. DLTs 1 90 40 3/3 2 140 40 4/3 3 210 40 4/4 4 210 60 10/10 1 (grade 3 elevated ALT) 5 140 75 7/6 1 (grade 3 drug hypersensitivity)

Non-DLT grade 3-4 treatment-related adverse events occurred in 11 pts, including nausea, cytopenia, hypotension, hypoalbuminemia, hypokalemia, elevated liver function tests (LFTs), and drug hypersensitivity. The maximum tolerated combination dose was not reached. However, the Compound A-2 dose escalation was stopped because pharmacokinetic (PK) exposures of Compound A-2 at the 140 mg/m² DL exceeded exposures previously shown in preclinical models, which resulted in robust target engagement and tumor regression in combination with CIS. There was no effect of CIS on the pharmacokinetics of Compound A-2. Compound A-2 terminal elimination half-life was about 16 hours and the Compound A-2 PK was proportional across the dosage ranges. RECIST partial responses were observed in 3 platinum-resistant/refractory pts (mesothelioma, ovarian, and triple-negative breast cancers) receiving Compound A-2 at 140 mg/m². Based on the above data, the recommended Phase II doses of Compound A-2 is 140 mg/m² and CIS is 75 mg/m² with RECIST antitumor responses observed in platinum-refractory pts.

FIG. 8 shows tumor response where maximum percent changes in target tumor diameter from baseline are depicted. Durations of progression free survival (PFS) in various cancers are shown in FIG. 9 .

As shown in FIG. 10 , partial response in the target region was observed in an ovarian cancer subject having BRCA2 germline mutation (W2626Q), where the patient received CIS 75 mg/m² on day 1 and Compound A-2 140 mg/m² on days 2 and 9 in cycle 1, and CIS 60 mg/m² in cycle 2 due to AE. Prior to the treatment with Compound A-2, the subject received the following treatments:

(i) debulking and paclitaxel with carboplatin;

(ii) carboplatin and doxorubicin: platinum resistant;

(iii) paclitaxel and bevacizumab; and

(iv) olaparib: no response.

As shown in FIG. 11 , partial response in the target region was observed in a triple-negative breast cancer subject having mutations in TP53 (R213*) and RB1 (deletion of exons 25-26), where the patient received CIS 75 mg/m² on day 1 and Compound A-2 140 mg/m² on days 2 and 9 in cycle 1. New brain metastases and leptomeningeal disease were discovered after completion of cycle 4, but no follow-up brain images are shown here as no brain imaging performed at the baseline. The subject Prior to the treatment with Compound A-2, the subject received the following treatments:

(i) mastectomy; doxorubicin, cyclophosphamide, paclitaxel; and radiation:

progressive disease PD) after 17 months;

(ii) re-excision; gemcitabine and ciaplatin: PD after 3 months; and

(iii)certain investigation agent: PD after 6 weeks.

Example 8. Phase I Trial of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 in Combination with Gemcitabine (Gem) in Patients (Pts) with Advanced Solid Tumors

In this Example, patients (pts), with advanced solid tumors measurable by RECIST 1.1 received IV a compound of Formula A-2 (Compound A-2) in combination with gem in a 3+3 dose-escalation design. Gem was administered on days 1 and 8 and Compound A-2 on days 2 and 9 of each 21-day cycle. Dose escalation was permitted if no dose-limiting toxicities (DLTs) were reported in a given treatment cycle. Fifty (50) pts (28 males and 22 females) of median age, 62 yrs (range 28-79 yrs), with Eastern Cooperative Oncology Group (ECOG) performance status 0/1:15/35 were treated. Primary tumors included non-small cell lung (NSCLC; n=6), pancreatic (n=2), breast (n=4), colorectal (n=15), head and neck (n=1), and other/missing (n=22). The following table describes the dosage levels (DLs) and various parameters for the various treatment cohorts.

Dose levels (DLs) were: Compound A-2 dose Gem dose No. pts DL (mg/m²) (mg/m²) treated 1 18 875 3 2 36 875 3 3 60 875 4 4 72 875 7 5 90 500 6 6 140 500 8 7 210 500 3 8 210 750 3 9 210 875 7 10 210 1000 6

Grade 3/4 treatment-related adverse events (AEs) occurred in 25 pts. DLTs occurred in 4 pts: 2 pts in DL4 (grade 3 thrombocytopenia; grade 3 elevated ALT and fatigue); 1 pt in DL5 (grade 3 elevated AST); 1 pt in DL6, (grade 3 elevated AST, ALT and grade 2 elevated ALP). The maximum tolerated dose was not reached but dose escalation was stopped at DL10 per investigator recommendation. The Compound A-2 exposure was relatively linear based on C_(max) and AUC_(0-∞) with the terminal elimination half-life of about 16 hours across DLs. No cumulative toxicity was observed. The best overall response was a partial response (PR) in 4 pts. The primary tumors in these four patients were breast cancer, head and neck cancer, NSCLC, and carcinoma of unknown primary origin. A pt with breast cancer in DL8 achieved PR at the 1^(st) assessment and the pt remains on trial >133 days. Two (2) pts had prolonged stable disease (SD) responses. The tumor response and duration of progression free survival (PFS) are shown in FIGS. 16 and 17 , respectively. For many patients, the duration of PFS extended beyond the 12-week primary endpoint. The median PFS was 8.3 weeks in patients treated at doses of Compound A-2<90 mg/m² in combination with gemcitabine 875 mg/m². Median PFS was 29.3 weeks in patients treated at doses of Compound A-2 ≥90 mg/m² in combination with gemcitabine 500 mg/m². Progression-free survival (PFS) of 415 days in 1 patient with renal cancer(papillary carcinoma) and >191 days (pt with GIST) were observed. Based on the above data, the recommended Phase II doses of Compound A-2 is 210 mg/m² and gem is 1000 mg/m².

Conclusion: Compound A-2 is well tolerated as monotherapy and in combination with CP, CIS, or gem with preliminary evidence of target modulation and antitumor activity. Compound A-2 will be further explored in early Phase II studies; in multiple tumor types, including triple-negative breast cancer and non-small cell lung cancer; and in patients with DDR aberrations.

Example 9: Phase I Dose Escalation Study of First-In-Class Ataxia Telangiectasia-Mutated and Rad3-Related (ATR) Inhibitor a Compound of Formula A-2 in Combination with Gemcitabine (Gem) and Cisplatin (CP) in Patients (Pts) with Advanced Solid Tumors

In this Example, patients (pts) with advanced solid tumors received IV a compound of Formula A-2 (Compound A-2) in combination with gem and CP in a 3+3 dose-escalation design. Compound A-2 was administered days 2, 9, and 16 every 3 weeks; gem was administered days 1 and 8 every 3 weeks, and CP was administered on day 1 every 3 weeks. The following table describes the dosage levels (DLs) and various parameters for the various treatment cohorts. The tumor response is shown in FIG. 16 .

Compound Gemcitabine Cisplatin No. of Patients No. of Patients A-2 Dose Dose Dose Treated/DLT with DLTs Cohort (mg/m²) (mg/m²) (mg/m²) Evaluable (DLT) 11 90 875 60 6/6 1 (thrombocytopenia/ neutropenia) 12 120 875 60 2/2 2 (febrile neutropenia/ thrombocytopenia, hypoxia/neutropenia)

Other Embodiments

While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds, methods, and processes of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example here. 

1-154. (canceled)
 155. A method of treating a proliferative disorder in a subject, the method comprising: administering to a subject in need thereof a DNA damaging agent and between about 12 and about 24 hours later administering to the subject a compound that inhibits ATR protein kinase; wherein the DNA-damaging agent is cisplatin; wherein the compound that inhibits ATR is represented by Formula A-3:

or a pharmaceutically acceptable salt thereof; and wherein the proliferative disorder is lung cancer.
 156. The method of claim 155, wherein the proliferative disorder is non-small cell lung cancer.
 157. The method of claim 155, wherein the proliferative disorder has one or more defects in the ATM signaling pathway.
 158. The method of claim 155, wherein the cancer has a defect in TP53.
 159. The method of claim 155, wherein the compound that inhibits ATR is administered about 16 hours after administration of the DNA damaging agent.
 160. A method of treating a proliferative disorder in a subject, the method comprising: administering to the subject in need thereof a platinating agent on day 1 and between about 12 and about 24 hours later administering to the subject a first dose of a compound that inhibits ATR protein kinase; and administering to the subject in need thereof a second dose of the compound that inhibits ATR protein kinase one week after administration of the first dose; wherein the platinating agent is cisplatin; wherein the compound that inhibits ATR is represented by Formula A-3:

or a pharmaceutically acceptable salt thereof; and wherein the proliferative disorder is lung cancer.
 161. The method of claim 160, wherein the lung cancer is non-small cell lung cancer.
 162. The method of claim 160, wherein the lung cancer has one or more defects in the ATM signaling cascade.
 163. The method of claim 160, wherein the lung cancer has a defect in TP53.
 164. The method of claim 160, wherein the compound that inhibits ATR is administered about 16 hours after administration of the DNA damaging agent.
 165. A method of treating a proliferative disorder in a subject, the method comprising: administering to the subject in need thereof gemcitabine and between about 12 and about 24 hours later administering to the subject a compound that inhibits ATR protein kinase; wherein the compound that inhibits ATR is represented by Formula A-3:

or a pharmaceutically acceptable salt thereof; wherein the proliferative disorder is pancreatic cancer.
 166. The method of claim 165, wherein the pancreatic cancer has a defect in the ATM signaling cascade.
 167. The method of claim 165, wherein the pancreatic cancer has a defect in TP53.
 168. The method of claim 165, wherein the compound that inhibits ATR is administered about 16 hours after administration of the DNA damaging agent.
 169. A method of treating a proliferative disorder in a subject, the method comprising: administering to the subject in need thereof gemcitabine on day 1 and between about 12 and about 24 hours later administering to the subject a first dose of a compound that inhibits ATR protein kinase; and administering to the subject in need thereof a second dose of the compound that inhibits ATR protein kinase every third day after administration of the first dose; wherein the compound that inhibits ATR is represented by Formula A-3:

or a pharmaceutically acceptable salt thereof; and wherein the proliferative disorder is pancreatic cancer.
 170. The method of claim 169, wherein the pancreatic cancer has one or more defects in the ATM signaling cascade.
 171. The method of claim 169, wherein the pancreatic cancer has a defect in TP53.
 172. The method of claim 169, wherein the compound that inhibits ATR is administered about 16 hours after administration of the DNA damaging agent. 